JP2023510194A - Novel anucleated cells and methods of their use for drug delivery - Google Patents

Abstract

Methods are provided for producing induced pluripotent stem cell-derived megakaryocytes and platelets. Such megakaryocytes or platelets may be genetically modified to contain a nucleic acid molecule encoding a therapeutic agent. The disclosure further provides methods and compositions for loading platelets or megakaryocytes with therapeutic agents and genetically altering platelets or megakaryocytes to express agents. The invention provides, for example, a composition comprising a population of induced pluripotent stem cell (iPSC)-derived platelets, wherein said platelets exhibit increased thrombin generation compared to a donor-derived platelet population having similar cell densities. To provide a composition shown.

Description

RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Patent Application No. 16/730,603, filed December 30, 2019, which application is hereby incorporated by reference in its entirety. .

FIELD The present disclosure relates to compositions of modified megakaryocyte progenitor cells, megakaryocytes, proplatelets, preplatelets and platelets for drug delivery, methods for producing the same, and the same. Regarding how to use .

Platelets are blood cells involved in clot formation and vascular repair at sites of active bleeding. Physiologically, platelets are produced in the bone marrow by parent cells called megakaryocytes (MKs), which constitute <0.1% of the cells in the bone marrow. Mature MKs are located outside the sinusoids in the bone marrow, with long structures called proplatelets extending into the circulation. Proplatelet precursors act as assembly lines for platelet production and sequentially release platelets from their ends.

Currently, platelets are obtained exclusively from human volunteer donors and deficiency is common. Extensive functional variability among donor platelets limits transfusion efficacy. The ever-growing demand for platelets exceeds the current supply by about 20% and the limited inventory of platelet units will be quickly depleted in an emergency.

Efficient delivery of therapeutic drugs to target sites is desirable to maximize therapeutic efficacy and minimize side effects. Although various nanoparticle-based approaches have been developed to improve tissue-targeted delivery of small molecules due to their enhanced permeability and retention (EPR) effects. , the nanoparticle approach has not been successful in packaging protein-based therapeutic agents, such as clotting factors or Ab drugs, due to their larger size. In addition, less than about 1% of the injected nanoparticles accumulate at most targeted sites, and repeated injections can lead to adverse immune responses to some components of nanoformulations (e.g., PEG), eroding efficacy. (Wilhelm Sea. Analysis of nanoparticle delivery to tumors. Nat Rev Mater. 2016; 1:16014). New methods to enhance existing physiological processes are urgently needed, particularly for protein-based therapeutics, to further enhance tissue-targeted drug delivery.

Methods for on-demand platelet production of defined platelet units to meet current and even planned platelet needs, as well as for the production of new vehicles for drug delivery is necessary.

Wilhelm Sea. Nat Rev Mater. (2016) 1:16014

In some aspects, the present disclosure is directed to compositions and methods of use of iPSC-derived preMKs, MKs, proplatelets, preplatelets and platelets for drug delivery.

In some aspects, a method for producing induced pluripotent stem cell (iPSC)-derived cells comprising a therapeutic agent, comprising differentiating pluripotent cells into hematopoietic endothelial cells in a first culture medium differentiating the hematopoietic endothelial cells into megakaryocyte progenitor cells in a second culture medium; differentiating the megakaryocyte progenitor cells into mature megakaryocytes; Methods are provided comprising differentiating under conditions, wherein one of the platelets, megakaryocytes, or megakaryocyte progenitor cells or combinations thereof comprises a therapeutic agent.

In some embodiments, megakaryocytes are CD42b+, CD61+, and DNA+. In some embodiments, platelets can be one or more of CD61+, DRAQ-, Calcein AM+, CD42a+, and CD62P+ in an activated state. Platelets need not express GPVI.

Platelets, megakaryocytes, or megakaryocyte progenitor cells may be loaded with therapeutic agents by, for example, receptor-mediated loading, passive loading, or covalent conjugation. Passive loading may involve incubating the therapeutic agent with a cell suspension comprising platelets, megakaryocytes, or megakaryocyte progenitor cells. Covalent conjugation may involve thiolation of membrane proteins with sulfhydryl-reactive cross-linkers, alkyne-reactive azides, high-affinity binders, and docking of antibodies to membrane-bound epitopes. Covalent conjugation may involve reacting an amine present in the therapeutic agent with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).

A therapeutic agent may be a chemokine, cytokine, growth factor, polypeptide, anti-angiogenic agent, polynucleotide, or small molecule. The anti-angiogenic agent may be doxorubicin, vincristine, irinotecan, or paclitaxel. It may be atezolizumab, ipilimumab, bevacizumab, cetuximab, or trastuzumab. The small molecule may be aripiprazole, esomeprazole, or rosuvastatin. Growth factors include platelet-derived growth factor isoforms (PDGF-AA, -AB and -BB), transforming growth factor-b (TGF-b), insulin-like growth factor-1 (IGF-1), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF or FGF-2), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF) , bone morphogenetic protein 2, -4 or -6 (BMP-2, -4, -6), von Willebrand factor, keratinocyte growth factor, FVII, FVIII, FIX, epidermal growth factor, or hair growth factor good too. The cytokine may be interleukin-1-beta, interleukin-2, or interleukin-12.

In some aspects, IPSC-derived cells, such as preMKs, MKs, proplatelets, preplatelets or platelets, are provided that comprise a therapeutic agent. In some embodiments, such cells may be loaded with or conjugated with a therapeutic agent. In some embodiments, such cells may be genetically engineered to express therapeutic agents. Also provided are compositions comprising IPSCc-derived cells, such as preMKs, MKs, platelet progenitors, preplatelets or platelets, comprising therapeutic agents. In some embodiments, a method of treating a subject comprising administering a therapeutically effective amount of a composition comprising IPSCc-derived cells, such as preMKs, MKs, proplatelets, preplatelets or platelets, comprising a therapeutic agent There is

One aspect of the present disclosure is a composition comprising a population of induced pluripotent stem cell (iPSC)-derived platelets, wherein the platelets exhibit increased thrombin generation compared to a donor-derived platelet population having similar cell densities. To provide a composition shown.

In some embodiments, thrombin generation in iPSC-derived platelets is two to three times greater than thrombin generation in a population of donor-derived platelets. In some embodiments, thrombin generation results in maximal thrombin concentrations of about 250 nM to about 850 nM. In some embodiments, thrombin generation results in maximal thrombin concentrations of about 450 to about 600 nM. In some embodiments, the lag time between stimulation and achieving maximum thrombin concentration in a population of iPSC-derived platelets is reduced compared to the lag time for a population of donor-derived platelets. The lag time for achieving maximum thrombin concentration in a population of induced pluripotent stem cell-derived platelets is about 7 to 15 minutes, where each population has about 0.5×10 ⁶ platelets/mL. to 3×10 ⁶ platelets/mL.

In some embodiments, the iPSC-derived platelet population comprises platelets that do not express glycoprotein VI. In some embodiments, the iPSC-derived platelet population has a CD61+, DRAQ-, calcein AM+, CD42a+, and CD62P- biomarker profile, a CD61+, DRAQ-, calcein AM+, CD42a+, and CD62P+ biomarker profile, and a CD61+ , including platelets having a biomarker profile selected from the group consisting of a CD62P+ biomarker profile. In some embodiments, greater than 70% of iPSC-derived platelets express CD61+, less than 10% of iPSC-derived platelets express CD42b, or less than 5% of iPSC-derived platelets express Fewer than 35% of platelets from iPSCs that express CD36 express calcein or a combination thereof. In some embodiments, the iPSC-derived platelet population has altered cell signaling compared to donor-derived platelets or megakaryocytes. In some embodiments, altered cell signaling comprises reduced CD62 activation following exposure to TRAP-6 or thrombin, or less than 15% of iPSC-derived platelets contain CD62p. In some embodiments, the composition further comprises thrombogenic microparticles such that the composition has a peak size of less than approximately 2 μm. In some embodiments, the thrombogenic microparticles range in size from 40 nm to 100 nm in diameter. In some embodiments, the thrombogenic microparticles constitute greater than 50% of the composition.

The present disclosure is described in the following detailed description with reference to several figures set forth as non-limiting examples of illustrative embodiments, where like reference numerals refer to several figures. Like parts are represented throughout the display.

FIG. 1 shows a global schematic for scalable differentiation of megakaryocyte progenitor cells (preMK), megakaryocytes (MK) and platelets (PLC) from iPSC lines. FIG. 2 shows an exemplary directed differentiation protocol from pluripotent stem cells to megakaryocytes in 2D, matrix-dependent systems such as cell culture plates or flasks. FIG. 3 is a schematic of an exemplary 3D matrix-independent method of directed differentiation using self-aggregating iPSC-derived spheroids in stirred tanks. FIG. 4 is a schematic diagram showing an exemplary directed differentiation protocol of iPSCs into megakaryocyte progenitor cells using a packed bed bioreactor strategy, a 3D matrix-dependent method. In the embodiments described herein, laminin 521 coated Raschig rings made of PTFE are used as macrocarriers to construct the packing layer. FIG. 5 shows the process of iPSC directed differentiation from hiPSCs to megakaryocytes and platelets. Cells can be loaded with small molecules, biologics, nucleotides, and other types of drug molecules at the pre-MK, MK, and PLC stages of differentiation. FIG. 6 is a schematic representation of a process for modifying platelets by covalent attachment of antibodies, in some embodiments for therapeutic effect. FIG. 7 shows the process of iPSC genetic engineering to express transgenes for the production of biological drugs followed by directed differentiation of hiPSCs into megakaryocytes and platelets. Viral transduction and other methods for transgene delivery into hiPSCs result in stable expression of biologics that can be used as drugs with efficient transcription and translation in pre-MKs, MKs, and PLCs can be used for Figures 8A-8C show expansion of induced pluripotent stem cells (iPSCs) with recombinant vitronectin using various growth media. Figure 8A shows iPSC growth in Essential 8 medium. FIG. 8B shows iPSC growth in StemFlex medium. FIG. 8C shows iPSC growth in Nutristem XF medium. Figures 9A-9C are flow charts evaluating the expression of pluripotency markers Tra-1-60, SSEA5, and differentiation marker SSEA1 in recombinant vitronectin-expanded iPSCs using various growth media. Cytometry data are shown. FIG. 9A shows pluripotent marker data from iPSCs expanded in Essential 8 medium. FIG. 9B shows pluripotent marker data from iPSCs expanded in StemFlex medium. FIG. 9C shows pluripotent marker data from iPSCs expanded in Nutristem XF medium. Figures 10A-10C show the results of a highly efficient single-cell passaging technique. FIG. 10A is a graph showing the growth reproducibility of the highly efficient single-cell passaging technique using a standardized plating density of 2×10 ⁴ cells/cm ² . P2, P4, and P5 refer to passages 2, 4, and 5. FIG. 10B is an image of cells thawed after cryogenic freezing, showing uniform undifferentiated morphology. FIG. 10C is a FACS analysis demonstrating >99% co-expression of SSEA-5 and TRA-1-60 cell surface markers in pluripotent cells prepared using a highly efficient single-cell passaging technique. be. Figures 11A-11C show expansion of iPSCs in self-aggregating spheroid cultures in 3D stirred tanks (no matrix). FIG. 11A shows microscopic images of iPSC spheroids in culture over time. FIG. 11B shows the increase in cell density over time in iPSC spheroid cultures. FIG. 11C shows average iPSC spheroid size over time in 3D culture. Figures 12A and 12B show pluripotency markers Tra-1-60, SSEA5, and differentiation marker SSEA1 in expanded iPSCs in self-aggregating spheroid cultures in 3D stirred tanks (no matrix). Flow cytometry data assessing the expression of . FIG. 12A shows pluripotent marker data for iPSCs after a single 7-day expansion in 3D stirred tanks. FIG. 12B shows pluripotency data of iPSCs after expansion for four consecutive 6-7 days in 3D stirred tanks. Figures 13A and 13B show iPSCs immunostained for the pluripotency factors Oct4 and Nanog and counterstained with nuclear dyes. FIG. 13A shows a portion of a 2D colony of iPSCs grown in vitronectin. FIG. 13B shows spheroids of iPSCs grown in 3D stirred conditions (no matrix). FIG. 14 shows karyotyping of metaphase chromosomes propagated from iPSCs grown in four consecutive 6-7 day expansions in 3D stirred tanks, demonstrating normal karyotypes after four 3D passages. do. FIG. 15 shows the morphological changes that occur over 6 days of stage 1 differentiation of iPSCs into hematopoietic endothelium on type IV collagen matrices in 2D culture vessels. Scale bar represents 100 μm. Figures 16A and 16B show representative stage 1 differentiation data of iPSC-derived cells. Figure 16A shows a representative flow cytometry analysis of iPSC-derived cells on day 6 of differentiation. Hematopoietic endothelial cells are identified through cell surface expression of CD31 and CD34. FIG. 16B shows the mean and range of stage 1 (day 6) differentiation efficiency across 41 independent iPSC directed differentiations. Figures 17A-17C show representative stage 2 data from iPSC differentiation cultures. FIG. 17A shows a stage 2 culture at day 6+6 with a hematopoietic endothelial (HE) monolayer in the background and megakaryocyte progenitor cells (preMK) released from the monolayer into suspension (arrows ). FIG. 17B shows flow cytometric analysis of stage 2 suspension cells, identifying CD43+ hematopoietic progenitor cells. FIG. 17C shows flow cytometry analysis of CD43+ hematopoietic cells, identifying CD43+CD41+CD14- megakaryocyte progenitor cells (preMK). Contaminating CD43+CD14+ myeloid progenitor cells are also identified in this analysis. Figures 17A-17C show representative stage 2 data from iPSC differentiation cultures. FIG. 17A shows a stage 2 culture at day 6+6 with a hematopoietic endothelial (HE) monolayer in the background and megakaryocyte progenitor cells (preMK) released from the monolayer into suspension (arrows ). FIG. 17B shows flow cytometric analysis of stage 2 suspension cells, identifying CD43+ hematopoietic progenitor cells. FIG. 17C shows flow cytometry analysis of CD43+ hematopoietic cells, identifying CD43+CD41+CD14- megakaryocyte progenitor cells (preMK). Contaminating CD43+CD14+ myeloid progenitor cells are also identified in this analysis. Figures 18A and 18B show the average compositional characteristics of stage 2 suspension cells. FIG. 18A shows the average daily purity of preMK released (ie % CD41+CD43+CD14− of viable suspension cells) over 10 days of Stage 2 release. FIG. 18B shows the median, quartiles, and range of contaminating myeloid progenitor cells (ie, % CD43+CD14+ of viable suspension cells) over stage 2 over 10 days. All cultures were initiated with iPSCs on type IV collagen matrices in 2D vessels. Data represent 41 independent differentiations. Figures 19A and 19B show the yield of released preMK. Figure 19A shows the average daily preMK released (ie viable CD41 + CD43 + CD14-) per 6-well equivalent (ie 2 ml medium, 9.5 ^cm surface area) during stage 2 directed differentiation cultures initiated with iPSCs. shows the yield of FIG. 19B shows the released preMK per 6-well equivalent (i.e., 2 ml medium, 9.5 cm ² surface area) between days 6+4 and 6+8 of stage 2 directed differentiation cultures initiated with iPSCs ( ie cumulative yield of viable CD41+CD43+CD14-). Each dot represents an independent iPSC directed differentiation culture on a type IV collagen matrix in a 2D culture vessel. 20A-20D show MK differentiation and platelet precursor production at stage 3. FIG. FIG. 20A shows iPSC-derived megakaryocyte progenitor cells at day 1 of stage 3 (top panel: high magnification; bottom panel: low magnification). FIG. 20B shows maturing megakaryocytes at day 2 of stage 3 (top panel: high magnification; bottom panel: low magnification). FIG. 20C shows mature megakaryocytes at day 4 of stage 3 (top panel: high magnification; bottom panel: low magnification). FIG. 20D illustrates spontaneous platelet precursor formation from mature iPSC-derived MKs after 4 days of stage 3 culture. Arrows indicate platelet precursors. A scale bar of 50 μm applies to all figures. Figures 21A-21C show representative flow cytometry analyzes from day 3 stage 3 cultures initiated from iPSCs. FIG. 21A identifies the CD61+ (megakaryocyte) fraction of stage 3 cells. FIG. 21B shows flow cytometric analysis of CD61+ megakaryocyte cells and identifies CD42a+CD42b+ mature MKs. Apoptotic CD42a+CD42b- cells can also be identified in this assay. FIG. 21C shows a subset breakdown of a representative stage 3 culture. Non-MKs are CD61-, immature MKs are CD61+CD42a-CD42b-, apoptotic MKs are CD61+CD42a+CD42b- and mature MKs are CD61+CD42a+CD42b+. Figures 22A and 22B show stage 0 and stage 1 differentiation initiated with self-aggregating spheroids of iPSCs. FIG. 22A begins with iPSCs dissociated into single cells on day −1, self-aggregated iPSC spheroids on day 0, partially differentiated spheroids on day 3, and hematopoietic endothelial cells on day 6. A series of photomicrographs of fully differentiated spheroids containing . Figure 22B shows day 6 flow cytometry data demonstrating successful CD31 + CD34 + hematopoietic endothelial differentiation using this approach. Figures 23A and 23B show that cultures expanded and harvested using the single-cell iPSC passaging approach were treated in 6-well ultra-low attachment plates using our 3D suspension differentiation method. It self-aggregates into 3D spheroids and can be effectively differentiated into preMK+ cells. FIG. 23A shows images of expanded cultures of single cells that aggregated (D0) and differentiated into hematopoietic endothelium (D6). FIG. 23B shows preMK+ production during stage 2 with single-cell passaged iPSCs (SC) compared to previous EDTA-passaged iPSC cultures (H). Figures 24A-24D describe stage 2 in directed differentiation initiated with iPSC self-aggregating spheroids. FIG. 24A shows self-aggregated iPSC-derived spheroids at day 6+4 during stage 2 of directed differentiation with preMKs (indicated by arrows) released from the spheroids into suspension. "HE" refers to hematopoietic endothelial monolayer. FIG. 24B shows flow cytometry analysis of harvested suspension cells stained for preMK markers CD41 and CD43. Figures 24C and 24D compare previous 2D data with data from two different 3D systems, ultra-low adhesion containers on orbital shakers and spinner flasks. FIG. 24C shows preMK purity over time in Stage 2. FIG. 24D shows preMK yield in stage 2 over time. Figures 24A-24D describe stage 2 in directed differentiation initiated with iPSC self-aggregating spheroids. FIG. 24A shows self-aggregated iPSC-derived spheroids at day 6+4 during stage 2 of directed differentiation with preMKs (indicated by arrows) released from the spheroids into suspension. "HE" refers to hematopoietic endothelial monolayer. FIG. 24B shows flow cytometry analysis of harvested suspension cells stained for preMK markers CD41 and CD43. Figures 24C and 24D compare previous 2D data with data from two different 3D systems, ultra-low adhesion containers on orbital shakers and spinner flasks. FIG. 24C shows preMK purity over time in Stage 2. FIG. 24D shows preMK yield in stage 2 over time. Figures 25A-25C show stage 3MK differentiation from 3D matrix-independent cultures initiated from self-aggregating spheroids of iPSCs. FIG. 25A shows a representative flow cytometric analysis from day 3 stage 3 cultures identifying the CD61+ (megakaryocyte) fraction of stage 3 cells followed by CD42a+CD42b+ mature MKs. Apoptotic CD42a+CD42b- cells can also be identified in this assay. FIG. 25B shows a subset breakdown of a representative stage 3 culture. Non-MKs are CD61-, immature MKs are CD61+CD42a-CD42b-, apoptotic MKs are CD61+CD42a+CD42b- and mature MKs are CD61+CD42a+CD42b+. FIG. 25C shows how mature MK fractions within day 3 stage 3 cultures are compared between 2D (matrix dependent) and 3D (matrix independent) approaches. Figures 25A-25C show stage 3MK differentiation from 3D matrix-independent cultures initiated from self-aggregating spheroids of iPSCs. FIG. 25A shows a representative flow cytometric analysis from day 3 stage 3 cultures identifying the CD61+ (megakaryocyte) fraction of stage 3 cells followed by CD42a+CD42b+ mature MKs. Apoptotic CD42a+CD42b- cells can also be identified in this assay. FIG. 25B shows a subset breakdown of a representative stage 3 culture. Non-MKs are CD61-, immature MKs are CD61+CD42a-CD42b-, apoptotic MKs are CD61+CD42a+CD42b- and mature MKs are CD61+CD42a+CD42b+. FIG. 25C shows how mature MK fractions within day 3 stage 3 cultures are compared between 2D (matrix dependent) and 3D (matrix independent) approaches. FIG. 26 shows proplatelet elongation of mature MKs recovered from 3D self-aggregating spheroid differentiation cultures. Examples of proplatelet elongation are indicated by arrows. Figures 27A-27D show increased preMK yield upon addition of soluble laminin 521 in two related 3D differentiation platforms. FIG. 27A. PreMK per well at day 6+6 of stage 2 with or without addition of laminin 521 during iPSC aggregation in StemFlex in ultra-low attachment U-bottom 96-well plates without agitation (day −1). indicate the number. FIG. 27B shows the per well per well at day 6+6 of stage 2 with or without addition of laminin 521 during the transition from stage 1 to stage 2 (day 6) in an ultra-low attachment U-bottom 96-well plate without agitation. Shows preMK numbers. FIG. 27C Laminin 521 during iPSC aggregation in StemFlex (day −1) or transition from stage 1 to stage 2 (day 6) in ultra-low attachment 6-well plates on an orbital shaker. Shown are preMK numbers per ml at day 6+6 in stage 2 with or without addition. FIG. 27D 6+5 days of stage 2 with or without addition of laminin 521 during single cell passage iPSC aggregation (day -1) in NutriStem in ultra-low attachment U-bottom 96-well plates without agitation. Shown are preMK numbers per ml in eyes. Figures 28A-28D show experiments performed to adjust the order and timing of the addition of the stage 1 media factors BMP4, bFGF and VEGFA. 28A is a schematic representation of the Stage 1 conditions tested in Experiment A. FIG. FIG. 28B shows the yield of stage 2 premK observed on day 6+4 of culture as described in panel A. FIG. 28C is a schematic representation of the Stage 1 conditions tested in Experiment B. FIG. FIG. 28D shows stage 2 preMK yields observed in the cultures described in panel C. FIG. The data in this figure suggest that stage 1 of differentiation can be effectively progressed using BMP4 alone for 24 hours followed by bFGF and VEGFA for 5 days before transitioning to stage 2 of differentiation. Figures 28A-28D show experiments performed to adjust the order and timing of the addition of the stage 1 media factors BMP4, bFGF and VEGFA. 28A is a schematic representation of the Stage 1 conditions tested in Experiment A. FIG. FIG. 28B shows the yield of stage 2 premK observed on day 6+4 of culture as described in panel A. FIG. 28C is a schematic representation of the Stage 1 conditions tested in Experiment B. FIG. FIG. 28D shows stage 2 preMK yields observed in the cultures described in panel C. FIG. The data in this figure suggest that stage 1 of differentiation can be effectively progressed using BMP4 alone for 24 hours followed by bFGF and VEGFA for 5 days before transitioning to stage 2 of differentiation. 29A-29C show immunofluorescence microscopy images of day 6 stage I cultures on laminin 521. FIG. FIG. 29A shows control cultures without WNT agonist. FIG. 29B shows cultures in which 0.6 μM WNT agonist CHIR98014 was added to differentiation cultures for the first 48 hours of Stage 1. FIG. FIG. 29C shows cultures in which 6 μM of WNT agonist CHIR99021 was added to differentiation cultures for the first 48 hours of Stage 1. FIG. 30A and 30B show immunofluorescence microscopy images of stage 2 cultures on laminin 521 at day 6+4. FIG. 27A shows control cultures without WNT agonist. FIG. 27B shows cultures supplemented with 0.6 μM WNT agonist CHIR98014 during the first 48 hours of Stage 1. FIG. FIG. 31 shows stage 1 differentiation of iPSCs on laminin 521-coated Raschig rings at days 3 and 6. FIG. Scale bar represents 500 μm. FIG. 32 shows stage 2 differentiation of iPSCs on laminin 521-coated Raschig rings at days 6+0 (100 μm scale bar) and 6+4 days (500 μm scale bar). Figures 33A-33C show flow cytometry data from stages of iPSC differentiation that proceed efficiently on Raschig ring supports. FIG. 33A shows stage 1 on day 6 with flow cytometry staining for hematopoietic endothelium markers CD31 and CD34. FIG. 33B shows stage 2 on day 6+2 with flow cytometry staining for megakaryocyte progenitor cell markers CD43 and CD41. FIG. 33C shows stage 3 on day 6+3+3 with flow cytometry staining for CD61 and CD42b. Figures 34A-34D show immunostained iPSC-derived megakaryocytes. FIG. 34A is a photomicrograph showing megakaryocytes immunostained for the megakaryocyte-specific protein β1-tubulin. FIG. 34B is a photomicrograph showing megakaryocyte nuclei visualized by nucleic acid staining. FIG. 34C is a photomicrograph showing the merged image of FIGS. 34A and 34B. FIG. 34D is a phase contrast image of immunostained megakaryocytes. For each figure, the scale bar represents 25 μm. Figures 35A-35F show immunostained iPSC-derived megakaryocytes. FIG. 35A is a photomicrograph showing iPSC-derived megakaryocytes immunostained for the α-granule-specific protein platelet factor 4 (PF4). FIG. 35B is a photomicrograph showing iPSC-derived megakaryocytes with immunostained nuclei. FIG. 35C is a photomicrograph showing iPSC-derived megakaryocytes immunostained for the α-granule-specific protein von Willebrand factor (VWF). FIG. 35D is a photomicrograph showing iPSC-derived megakaryocytes immunostained for CD61, a megakaryocyte-specific cell surface marker. FIG. 35E is a photomicrograph showing the superimposed images of FIGS. 35A, 35B, and 35C. FIG. 35F is a photomicrograph showing the superimposed images of FIGS. 35A, 35B, and 35D. For each figure, the scale bar represents 25 μm. Figures 36A-36F show immunostained iPSC-derived megakaryocytes. FIG. 36A is a photomicrograph showing iPSC-derived megakaryocytes immunostained for LAMP1, a dense granule-specific protein. FIG. 36B is a photomicrograph showing iPSC-derived megakaryocytes with immunostained nuclei. FIG. 36C is a photomicrograph showing iPSC-derived megakaryocytes immunostained for serotonin, a dense granule-specific protein. FIG. 36D is a photomicrograph showing iPSC-derived megakaryocytes immunostained for CD61, a megakaryocyte-specific cell surface marker. FIG. 36E is a photomicrograph showing the superimposed images of FIGS. 36A, 36B, and 36C. FIG. 36F is a photomicrograph showing the superimposed images of FIGS. 36A, 36B, and 36D. For each figure, the scale bar represents 25 μm. Figures 37A-37D are electron microscope images showing iPSC-derived megakaryocytes. FIG. 37A is an electron microscope image showing iPSC-derived megakaryocytes producing microparticles (see, eg, arrows). FIG. 37B is an electron microscope image showing iPSC-derived megakaryocytes and multivesicular bodies (arrows; enlarged in inset). FIG. 37C is an electron microscope image showing an iPSC-derived megakaryocyte characterized by polymorphonuclei, glycogen granules, alpha granules, and an invaginated membrane system. FIG. 37D is an electron microscope image showing the endoplasmic reticulum and mitochondria of iPSC-derived megakaryocytes. Figures 38A and 38B illustrate characteristic gene expression changes that occur during directed differentiation of iPSCs to megakaryocytes. For all expression analyses, expression in iPSCs was set to 1 and all other expression values are expressed relatively. FIG. 38A. Pluripotency-associated genes in iPSCs, day 6 cells (end of stage 1), days 6+4 and 6+5 (stage 2), and days 6+5+1 to 6+5+4 (stage 3). Figure 2 illustrates relative gene expression of Oct4. Figure 38B shows iPSCs, day 6 cells (end of stage 1), days 6+4 and 6+5 (stage 2), and days 6+5+1 to 6+5+4 (stage 3), important for megakaryocyte maturation. Figure 2 illustrates relative gene expression of the transcription factor NFE2. FIG. 39 shows OCT4, SOX2, NANOG, and ZFP42 downregulated during differentiation, ZFPM1, NFE2, RUNX1, MEIS1, and GATA1 upregulated during differentiation, and PBX1 and PBX1 remaining at substantially consistent levels. FIG. 4 is a heat map showing MYC; FIG. Figures 40A-40C provide the size distribution of iPSC-derived megakaryocytes. FIG. 40A shows a representative example of β1-tubulin staining of iPSC-derived megakaryocytes utilized to collect size measurements of iPSC-MKs and compare them with MKs from other sources. FIG. 40B shows the size distribution of iPSC-derived megakaryocytes, including median, quartile, and range. FIG. 40C compares the size distribution data of iPSC-derived MKs with megakaryocytes derived from different bone marrow sources. Figures 41A and 41B provide polyploidy measurements for iPSC-derived megakaryocytes. FIG. 41A shows representative examples of DNA ploidy measurements performed on iPSC-derived megakaryocytes. FIG. 41B compares DNA ploidy measurements of iPSC-MKs with MKs from other sources. FIG. 42 provides a comparison of the presence or absence and concentration ranges of various factors in hiPSC-MK lysates of megakaryocytes obtained by the methods of the present disclosure and certain controls. '*' denotes proteins measured in hiPSC-MKs that have not been previously described in megakaryocytes or platelets, and '¥' denotes inflammatory cytokines. Figures 43A and 43B show hiPSC platelet production. Figure 43A shows flow cytometric analysis of anucleated and nucleated cells (top, left). Nucleated cells contained numerous CD41 ⁺ CD42 ⁺ megakaryocytes (top, right). Anucleated cells positive for CD41 ⁺ , CD42 ⁺ , and Calcein AM were evaluated using flow cytometry and platelets were gated by size (1-5 microns). FIG. 43B is a graph showing the cumulative yield of CD41 ⁺ CD42 ⁺ Calcein AM ⁺ platelet-sized particles per well during Stage 3 of the directed differentiation protocol described herein. Figures 44A and 44B show resting and activated platelets. FIG. 44A contains images of platelets recovered from megakaryocyte cultures assessed by electron microscopy. FIG. 44B shows that β1-tubulin coils and CD61 expression are characteristic features of resting platelets, and that lamellipodia and filopodia are activated, as revealed by F-actin staining. Includes photomicrographs showing that it is characteristic of platelets that have been plated. Figure 45 is a complete panel of immunophenotyping characterization of human iPSC-derived platelets. The top right panel shows the forward scatter versus side scatter profile of the cells. These cells are then gated with DRAQ- (upper middle panel), which indicates a lack of genomic material and the platelet surface marker CD61 (upper right panel), before being evaluated for multiple parameters. Gated cells were re-evaluated for forward scatter versus side scatter parameters (bottom left panel). These were also stained with Calcein AM to assess viability (second panel below). Gated cells were further investigated for CD42a expression (third bottom panel) and for common platelet surface markers. These were also stained for CD62P, an indicator of activation (bottom right panel), providing evidence that these platelets were quiescent. Figures 46A-46C show the relative lack of glycoprotein VI (GPVI) in hiPSC platelet populations compared to those from human whole blood. FIG. 46A shows FSC vs. SSC parameters elucidating similar size (FSC) and granularity (SSC) characteristics between hiPSC platelets and donor-derived human platelets. FIG. 46B shows the abundance of CD61 expression on both hiPSC platelets and donor-derived human platelets. FIG. 46C demonstrates that CD61+ hiPSC platelet populations are mostly devoid of GPVI on their cell surface, whereas donor-derived human platelets are almost exclusively GPVI-positive. Figure 47 is a graph of thrombin generation by human iPSC-derived platelets in aqueous buffer and primary platelets in plasma over time when treated with recombinant human tissue factor. Figures 48A and 48B are in vivo photomicrographs demonstrating that human iPSC-derived platelets are incorporated into thrombi using a laser injury model for clot formation in mouse cremaster arterioles. FIG. 48A shows human donor platelets (indicated by the dark circled area within the dotted box) incorporated into the thrombus after laser injury was performed and by proximal infusion of platelets near the injury site. FIG. 48B shows human iPSC-derived platelets incorporated into a thrombus (dark circled area in dotted box) using the same laser injury model. Figures 49A-49I show antibody loading on washed donor-derived human platelets. Figure 49A shows the isotype antibody signals that define the gating strategy for this figure. FIG. 49B shows that washed donor-derived human platelets are almost exclusively CD61+ when stained exclusively for CD61. FIG. 49C shows the expression of both CD61 and Cy5.5 conjugated to human IgG antibody after co-incubation in platelet suspension. Figure 49D shows human IgG in antibody-loaded donor-derived human platelets after washing the IgG-loaded donor-derived human platelets with phosphate-buffered saline (PBS), centrifugation, and resuspension. Co-expression of CD61 and Cy5.5 conjugated to antibodies is shown. FIG. 49E is a photograph of platelet pellets with and without human IgG Cy5.5 loaded by co-incubation of antibody and platelet suspension. Cy5.5 dye is visible to the naked eye. FIG. 49F shows a dose-dependent increase in human IgG Cy5.5 uptake into washed human platelets. Figure 49G is a line graph plotting the geometric mean of the fluorescence signals shown in Figure 49F. Figure 49H depicts human entrapped in donor-derived human platelets by microplate reader measurement of human IgG Cy5.5 signal from platelet lysates and calculated from a standard curve generated from free Cy5.5 dye. IgG quantification is shown. FIG. 49I shows a similar dose-dependent escalation in antibody loading of donor-derived human platelets using the Dylight-498 conjugated version of the FDA-approved antibody drug atezolizumab. Figures 49A-49I show antibody loading on washed donor-derived human platelets. Figure 49A shows the isotype antibody signals that define the gating strategy for this figure. FIG. 49B shows that washed donor-derived human platelets are almost exclusively CD61+ when stained exclusively for CD61. FIG. 49C shows the expression of both CD61 and Cy5.5 conjugated to human IgG antibody after co-incubation in platelet suspension. Figure 49D shows human IgG in antibody-loaded donor-derived human platelets after washing the IgG-loaded donor-derived human platelets with phosphate-buffered saline (PBS), centrifugation, and resuspension. Co-expression of CD61 and Cy5.5 conjugated to antibodies is shown. FIG. 49E is a photograph of platelet pellets with and without human IgG Cy5.5 loaded by co-incubation of antibody and platelet suspension. Cy5.5 dye is visible to the naked eye. FIG. 49F shows a dose-dependent increase in human IgG Cy5.5 uptake into washed human platelets. Figure 49G is a line graph plotting the geometric mean of the fluorescence signals shown in Figure 49F. Figure 49H depicts human entrapped in donor-derived human platelets by microplate reader measurement of human IgG Cy5.5 signal from platelet lysates and calculated from a standard curve generated from free Cy5.5 dye. IgG quantification is shown. FIG. 49I shows a similar dose-dependent escalation in antibody loading of donor-derived human platelets using the Dylight-498 conjugated version of the FDA-approved antibody drug atezolizumab. Figures 50A and 50B demonstrate direct loading of atezolizumab onto washed human PLC. FIG. 50A includes representative photomicrographs visualizing uptake of atezolizumab with AlexaFluor488 anti-human IgG in addition to appropriate controls (IgG only). FIG. 50B includes micrographs captured at higher magnification to demonstrate intracellular atezolizumab in human donor platelets and internalization of atezolizumab into human donor platelets. Figures 51A and 51B demonstrate passive loading of ipilimumab on human iPSC-derived platelets. FIG. 51A is a flow cytometry histogram plot showing the dose-dependent increase in antibody signal with increasing amounts of ipilimumab in reaction vessels containing platelet suspensions. FIG. 51B is an immunofluorescence micrograph showing ipilimumab both in and on human iPSC-derived platelets observed with antibody staining for CD61 (surface marker) and platelet factor 4, or the granule marker PF4. Figures 52A-52F describe passive drug loading in CD34+ mobilized peripheral blood-derived platelets. FIG. 52A is a flow cytometry dot plot displaying cells as events plotted by forward scatter (size) versus side scatter (granularity). Figure 52B is a flow cytometry dot plot showing a CD61+ cell population of approximately 90% of CD34+ mobilized peripheral blood-derived platelets. FIG. 52C is a flow cytometry histogram plot showing a dose-dependent increase in Cy5.5-conjugated ipilimumab binding to CD34+ mobilized peripheral blood-derived platelets. FIG. 52D shows the dose-dependent increase in mean fluorescence intensity (geometric mean ) is plotted. FIG. 52E is a bar graph that is a quantification of ipilimumab binding to CD34+ mobilized peripheral blood-derived platelets. FIG. 52F includes photomicrographs showing the distribution of ipilimumab on CD61+ and PF4 (platelet factor 4)+ platelets. Figures 53A-53D show uptake and release of atezolizumab by platelets upon thrombin activation. Figures 53A and 53B are histograms of flow cytometry analysis showing platelet uptake/release of atezolizumab or ipilimumab upon activation with thrombin. FIG. 53C is a representative photomicrograph of atezolizumab (25 μg/ml) of resting platelets. FIG. 53D is a representative photomicrograph of platelets loaded with atezolizumab and activated. Figures 54A-54E illustrate covalent conjugation of human IgGl on washed human platelets. FIG. 54A provides an example of modifying surface proteins on platelet membranes by converting primary amines to reactive sulfhydryls. Figure 54B provides an example of linking a maleimide to a protein. FIG. 54C provides an example of reacting maleimide-linked proteins with platelets with exposed sulfhydryl groups to form thioether bonds. FIG. 54D shows increased IgG conjugation to washed donor-derived human platelet preparations in response to platelets exposed to fixed but increasing doses of Traut's reagent to render the surface more reactive. 3 is a flow cytometry histogram plot showing . Figure 54E shows a fixed amount of IgG conjugation to washed donor-derived human platelets in the absence of maleimide (SMCC) conjugation to IgG, thus reducing the affinity of the antibody to reactive sulfhydryls on the platelet membrane. Flow cytometry histograms showing gating. Figures 54A-54E illustrate covalent conjugation of human IgGl on washed human platelets. FIG. 54A provides an example of modifying surface proteins on platelet membranes by converting primary amines to reactive sulfhydryls. Figure 54B provides an example of linking a maleimide to a protein. FIG. 54C provides an example of reacting maleimide-linked proteins with platelets with exposed sulfhydryl groups to form thioether bonds. FIG. 54D shows increased IgG conjugation to washed donor-derived human platelet preparations in response to platelets exposed to fixed but increasing doses of Traut's reagent to render the surface more reactive. 3 is a flow cytometry histogram plot showing . Figure 54E shows a fixed amount of IgG conjugation to washed donor-derived human platelets in the absence of maleimide (SMCC) conjugation to IgG, thus reducing the affinity of the antibody to reactive sulfhydryls on the platelet membrane. Flow cytometry histograms showing gating. Figures 55A-55C use the covalent conjugation strategy outlined in Figure 53 for conjugating a molecule, in this example the antibody drug ipilimumab, to human iPSC-derived platelets in one repeated application. Provide an example. Figure 55A shows that human iPSC-derived platelets are >89% CD61+, confirming that they express platelet-specific surface markers. FIG. 55B is a flow cytometry histogram plot showing efficient covalent conjugation that occurs when ipilimumab reacts with SMCC (maleimide) and platelets are treated with Traut's reagent to facilitate covalent ligation. FIG. 55C is a photomicrograph showing the staining pattern of ipilimumab for CD61 and PF4 in human resting iPSC-derived platelets. Figures 56A-56C demonstrate passive loading of megakaryocytes. FIG. 56A shows iPSC-derived megakaryocytes and platelets incubated in the presence of dyelight488-labeled atezolizumab (ie, anti-PDL1). To demonstrate mature megakaryocytes, cells were reverse labeled with CD61. FIG. 56B shows subcellular localization of Dylight488 atezolizumab in megakaryocytes and platelets. FIG. 56C further demonstrates subcellular localization of platelet factor-4 pigment to alpha granules. Figures 57A-57D demonstrate biological drug loading in human iPSC-derived megakaryocyte progenitors. Figure 57A shows CD61 staining in this population, which can be positively identified as preMK. FIG. 57B shows fibrinogen uptake staining alpha granules. FIG. 57C shows the signal from the secondary antibody detecting ipilimumab uptake. FIG. 57D is a merged image demonstrating co-localized cells with both surface (CD61) and granular (fibrinogen) staining with observed ipilimumab uptake. Figures 58A-58D demonstrate covalent conjugation of ipilimumab to megakaryocyte precursors. Figure 58A shows CD41 expression of cells at the end of stage 2, identifying them as megakaryocyte progenitors by flow cytometry. FIG. 58B shows a CD43+CD41+ cell further characterizing it as a megakaryocyte progenitor by flow cytometry. Figure 58C is a flow cytometry dot plot in untreated conditions with detection (secondary) antibody. Figure 58D is a flow cytometry dot plot showing robust expression of ipilimumab compared to controls. Figures 59A-59D demonstrate covalent conjugation of ipilimumab to human iPSC-derived megakaryocytes. Figure 59A is a flow cytometry dot plot demonstrating robust expression of CD61 as part of a gating strategy to identify megakaryocytes. FIG. 59B is a flow cytometry dot plot showing the CD42a+CD61+ population in these cultures with a further refined gating strategy to identify megakaryocytes. FIG. 59C is a flow cytometry dot plot in untreated conditions with detection (secondary) antibody. FIG. 59D is a flow cytometry dot plot demonstrating efficient loading of ipilimumab in contrast to controls. Figures 60A and 60B show the efficient loading of washed donor-derived human platelets with the small molecule chemotherapeutic drug doxorubicin. FIG. 60A is a flow cytometry histogram plot showing retention of doxorubicin in washed platelets after multiple washing steps. FIG. 60B is a flow cytometry histogram plot showing doxorubicin detected in platelets after co-incubation at various time points in minidialysis cassettes using constant agitation at room temperature on an orbital shaker. FIG. 61 includes photomicrographs showing mock transduced preMKs and preMKs transduced with a lentiviral vector containing a nucleic acid molecule encoding the EF1 alpha promoter driving expression of ZsGreen fluorescent protein. Figures 62A-62C show retention of genetic alterations introduced into premegakaryooctyes via lentiviral transduction. FIG. 62A. Mock transduced megakaryocyte progenitor-derived platelet cells and transduced with a lentiviral vector (lenti(+)poly) encoding the EF1 alpha promoter driving expression of ZsGreen fluorescent protein together with polybrene. Includes brightfield and fluorescence micrographs of platelets derived from megakaryocyte progenitors. FIG. 62B Flow cytometry showing CD61+ platelets from mock transduced megakaryocytes and megakaryocytes transduced with a lentiviral vector encoding the EF1 alpha promoter driving expression of ZsGreen fluorescent protein with polybrene. Contains metric data. FIG. 62C is a flow cytometry histogram plot further illustrating the ZsGreen signal detected in lentiviral transduction conditions as opposed to mock transduction controls. Figures 62A-62C show retention of genetic alterations introduced into premegakaryooctyes via lentiviral transduction. FIG. 62A. Mock transduced megakaryocyte progenitor-derived platelet cells and transduced with a lentiviral vector (lenti(+)poly) encoding the EF1 alpha promoter driving expression of ZsGreen fluorescent protein together with polybrene. Includes brightfield and fluorescence micrographs of platelets derived from megakaryocyte progenitors. FIG. 62B Flow cytometry showing CD61+ platelets from mock transduced megakaryocytes and megakaryocytes transduced with a lentiviral vector encoding the EF1 alpha promoter driving expression of ZsGreen fluorescent protein with polybrene. Contains metric data. FIG. 62C is a flow cytometry histogram plot further illustrating the ZsGreen signal detected in lentiviral transduction conditions as opposed to mock transduction controls. Figures 63A-63D describe a bioreactor for platelet production. FIG. 63A is an image of a platelet bioreactor. Figure 63B is a cross-sectional view of a platelet bioreactor. FIG. 63C is a top view of the platelet bioreactor. Figure 63D is a schematic of a platelet bioreactor. Figure 64 is a schematic side view of a bioreactor for platelet production with upper and lower channels separated by a porous membrane (dotted line). Megakaryocytes trapped on the porous membrane in the first channel are subjected to shear stress and extend processes that release platelets (dark circles) into the channel below. Straight arrows indicate fluid flow. FIG. 65 is a microscopic image of MKs under physiological shear illustrating proplatelet production when trapped on a porous membrane. Figures 66A-66E illustrate that genetic engineering can lead to functional expression of fluorescent proteins in PLC. FIG. 66A is a diagram illustrating the progression of cell types from pluripotent stem cells to platelets (PLC). FIG. 66B is a fluorescence microscope image showing transduced iPSC-derived ZsGreen+ cells. FIG. 66C is a graph showing ZsGreen expression retained in cells during different time points in stage 2 of differentiation. FIG. 66D is a graph showing ZsGreen expression retained in megakaryocytes. FIG. 66E is a graph showing preserved ZsGreen expression in CD61+ PLCs. Figures 66A-66E illustrate that genetic engineering can lead to functional expression of fluorescent proteins in PLC. FIG. 66A is a diagram illustrating the progression of cell types from pluripotent stem cells to platelets (PLC). FIG. 66B is a fluorescence microscope image showing transduced iPSC-derived ZsGreen+ cells. FIG. 66C is a graph showing ZsGreen expression retained in cells during different time points in stage 2 of differentiation. FIG. 66D is a graph showing ZsGreen expression retained in megakaryocytes. FIG. 66E is a graph showing preserved ZsGreen expression in CD61+ PLCs. Figures 66A-66E illustrate that genetic engineering can lead to functional expression of fluorescent proteins in PLC. FIG. 66A is a diagram illustrating the progression of cell types from pluripotent stem cells to platelets (PLC). FIG. 66B is a fluorescence microscope image showing transduced iPSC-derived ZsGreen+ cells. FIG. 66C is a graph showing ZsGreen expression retained in cells during different time points in stage 2 of differentiation. FIG. 66D is a graph showing ZsGreen expression retained in megakaryocytes. FIG. 66E is a graph showing preserved ZsGreen expression in CD61+ PLCs. FIG. 67 depicts representative flow cytometry scatter plots from freshly isolated donor platelets and PLCs produced by the millifluidic bioreactor. FIG. 68 depicts representative flow cytometry scatter plots from freshly isolated normal adult donor platelets and PLCs produced by the Millifluidics bioreactor. FIG. 69 depicts the difference in cell surface expression of CD42b and CD36 between freshly isolated normal adult donor platelets and PLCs produced by the Millifluidics bioreactor. Figures 70A and 70B compare the platelet activation marker CD62p after exposure to thrombin receptor activator peptide (TRAP-6). FIG. 70A depicts flow cytometry scatter plots showing the percentage of CD62p+ cells in resting and TRAP-6-activated donor-derived iPSCs and iPSC-derived platelets. FIG. 70B is a graph illustrating the results observed in FIG. 70A. Figures 70A and 70B compare the platelet activation marker CD62p after exposure to thrombin receptor activator peptide (TRAP-6). FIG. 70A depicts flow cytometry scatter plots showing the percentage of CD62p+ cells in resting and TRAP-6-activated donor-derived iPSCs and iPSC-derived platelets. FIG. 70B is a graph illustrating the results observed in FIG. 70A. Figure 71 compares the platelet activation marker CD62p after exposure to thrombin (1 U/ml). FIG. 72 depicts a representative histogram generated using the Multisizer device. Figures 73A and 73B compare representative forward and side scatter plots of donor-derived and iPSC-derived platelets. FIG. 73A shows representative forward and side scatter plots of donor-derived platelets. FIG. 73B shows representative forward and side scatter plots of iPSC-derived platelets. Figures 73A and 73B compare representative forward and side scatter plots of donor-derived and iPSC-derived platelets. FIG. 73A shows representative forward and side scatter plots of donor-derived platelets. FIG. 73B shows representative forward and side scatter plots of iPSC-derived platelets. Figures 74A and 74B compare the morphology of donor-derived and PLC platelets by donor electron microscopy. FIG. 74A is an electron microscopy image showing donor-derived platelets. Scale bar represents 800 nm. FIG. 74B is an electron microscopy image showing the PLC. Scale bar represents 600 nm. Figure 75 shows microvesicles derived from iPSC-derived MKs and within the PLC population. They are heterogeneous in size ranging from less than 40 nm to over 1000 nm in diameter. Figure 76 shows the reproducibility of thrombin generation in PLC compared to donor platelets. Each group represents three independent samples monitored for thrombin generation as described in Methods. FIG. 77 depicts representative dose and response curves for PLC in thrombin generation. Figures 78A and 78B depict the contribution of microvesicles to thrombin generation within the PLC. As shown in Figure 76, PLC showed a rapid and strong burst of thrombin generation unlike that of fresh donor platelets. FIG. 78A is a graph depicting donor-derived platelets and PLCs that were subjected to a 1 μm filter to remove all cellular material larger than 1 μm. FIG. 78B shows that filtered PLC exhibited thrombogenic activity leading to thrombin levels similar to those of donor platelets. Figures 78A and 78B depict the contribution of microvesicles to thrombin generation within the PLC. As shown in Figure 76, PLC showed a rapid and strong burst of thrombin generation unlike that of fresh donor platelets. FIG. 78A is a graph depicting donor-derived platelets and PLCs that were subjected to a 1 μm filter to remove all cellular material larger than 1 μm. FIG. 78B shows that filtered PLC exhibited thrombogenic activity leading to thrombin levels similar to those of donor platelets.

While the figures identified above describe embodiments of the present disclosure, other embodiments are contemplated as described in the discussion. This disclosure presents illustrative embodiments by way of representation and not limitation. Those skilled in the art can devise numerous other modifications and embodiments that fall within the scope and spirit of the principles of the disclosed embodiments.

DETAILED DESCRIPTION Definitions Unless otherwise defined, all scientific and technical terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this disclosure: Singleton et al. , Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); , R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below unless specified otherwise.

The terms "agent," "therapeutic agent," "therapeutic composition," "drug," or "therapeutic agent" can be used interchangeably and refer to any small molecule compound, antibody, nucleic acid molecule, or It is meant to include polypeptides, or fragments thereof.

The term "antibody," as used herein, refers to an immunoglobulin molecule that specifically binds an antigen. The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic determining variable regions of the intact antibody.

"Modify" or "change" means increase or decrease. The change is only 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or 40%, 50%, 60%, or even 70%, 75% %, 80%, 90%, or even 100%.

"Biological sample" means any tissue, cell, fluid, or other material derived from an organism.

"Capture reagent" means a reagent that specifically binds to a nucleic acid molecule or polypeptide to select or isolate the nucleic acid molecule or polypeptide.

By "cell composition" is meant any composition comprising one or more isolated cells.

By "cell viability" is meant cell viability.

As used herein, "clinical grade" means a cell or cell line that has been derived or obtained using current pharmaceutical manufacturing and quality control regulations (GMP) permitting clinical use in humans. shall refer to GMP is a quality assurance system used in the pharmaceutical industry to ensure that the final product meets preset specifications. GMP extends to both manufacturing and testing of final products. GMP not only requires traceability of raw materials, but also that production is in accordance with validated Standard Operating Procedures (SOPs).

By "detectable level" is meant that the amount of analyte is sufficient for detection using methods routinely used to perform such analyses.

"Detect" refers to identifying the presence, absence or amount of an analyte to be detected.

The term "covalent conjugation" refers to the use of chemical linkers that react with specific chemical groups on the molecule to be conjugated. In some embodiments, the covalent conjugation of the therapeutic composition to another molecule or compound connects the amine on the therapeutic composition to the linker succinimidyl 4-(N-maleimidomethyl)cyclohexane-1. - achieved by reacting with a carboxylate (SMCC).

A "detectable label" is one that, when attached to a molecule of interest, renders the molecule detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. means a composition that For example, useful labels include radioisotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (eg, commonly used in ELISA), biotin, digoxigenin, or haptens. .

By "disease" is meant any condition or disorder that damages or interferes with the normal functioning of cells, tissues, or organs. Examples of diseases include ischemic injuries such as stroke, myocardial infarction, or any other ischemic event that causes tissue damage, peripheral vascular disease, wounds, burns, fractures, blunt trauma, arthritis, and inflammatory disease. Any disease or injury that results in a reduction in cell number or biological function, including.

By "effective amount" is meant the amount of agent necessary to produce an intended effect.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. Preferably, the portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of the reference nucleic acid molecule or polypeptide. Fragments may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

The terms "isolated," "purified," or "biologically pure" mean that the material is free to varying degrees of components that normally accompany it when found in its native state. point to "Isolate" indicates some degree of separation from the original source or surroundings. "Purity" indicates a higher degree of separation than isolation. A "purified" or "biologically pure" protein is defined as being sufficiently isolated so that any impurities do not substantially affect the biological properties of the protein or cause other detrimental consequences. Does not contain materials. That is, the nucleic acids or peptides of the present disclosure may be derived from cellular material, viral material, or culture medium if the nucleic acid or peptide was produced by recombinant DNA techniques, or chemical precursors or peptides if chemically synthesized. It is purified if it is substantially free of other chemicals. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can indicate that a nucleic acid or protein gives rise to essentially one band in an electrophoresis gel. For proteins that can be subjected to modifications, eg, phosphorylation or glycosylation, the different modifications can give rise to different isolated proteins, which can be purified separately.

By "isolated polynucleotide" is meant a nucleic acid (eg, DNA) that is free of the genes that flank the gene in the naturally occurring genome of the organism from which the nucleic acid molecule of the disclosure is derived. Thus, the term exists, for example, in a vector; in an autonomously replicating plasmid or virus; or in a recombinant DNA that is incorporated within prokaryotic or eukaryotic genomic DNA; or as a separate molecule independent of other sequences. (eg, cDNA or genomic or cDNA fragments generated by PCR or restriction endonuclease digestion). In addition, the term includes RNA molecules transcribed from DNA molecules, as well as recombinant DNAs that are part of a hybrid gene encoding additional polypeptide sequence.

By "isolated polypeptide" is meant a polypeptide of this disclosure that is separated from the components that naturally accompany it. Generally, a polypeptide is isolated if it is at least 60% by weight free of naturally associated proteins and naturally occurring organic molecules. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of a polypeptide of the disclosure. An isolated polypeptide of the present disclosure can be obtained, for example, by extraction from natural sources, by expression of recombinant nucleic acid encoding such a polypeptide, or by chemical synthesis of the protein. Purity can be measured by any reasonable method, such as column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

The term "hematopoietic endothelial cell" as used herein refers to a cell capable of differentiating to give rise to a hematopoietic or endothelial cell type, which is optionally pluripotent. It may be derived from stem cells. Hematopoietic endothelial cells are normally adherent to extracellular matrix proteins and/or other hematopoietic endothelial cells and can be characterized by expression of the markers CD31 and CD34.

By "marker" is meant any protein or other epitope that has a change in expression level or activity associated with a characteristic or condition.

The term “megakaryocyte” (MK), as used herein, refers to a large (eg, ≧10 μm diameter) polyploid hematopoietic cell that has the property of giving rise to platelet precursors and/or platelets. One morphological feature of mature megakaryocytes is the development of large polymorphonuclei. Mature megakaryocytes may stop proliferating but continue to increase in DNA content through endomitosis, with a parallel increase in cell size.

The term "megakaryocyte progenitor cell" (preMK), as used herein, refers to a mononuclear hematopoietic cell that is committed to the megakaryocyte lineage and is the precursor of mature megakaryocytes. Megakaryocyte progenitor cells are normally found (but not limited to) in the bone marrow and other hematopoietic sites, but are also derived from pluripotent stem cells, as well as hematopoietic endothelial cells that are themselves derived from pluripotent stem cells. It can be generated by differentiation and the like.

The term "microparticle" refers to very small (<1 micron) phospholipid vesicles that are shed from megakaryocytes or other cells. Microparticles may contain genetic material such as RNA and may express extracellular markers of their parental cells. Megakaryocyte- and platelet-derived microparticles may have roles in numerous pathways, including hemostasis and inflammation.

"Passive drug loading" means uptake of a therapeutic composition by cells (eg, platelets or their precursors) without conjugation or mechanical or chemical disruption or modification of the cells. For example, liposomal delivery systems can be used for passive drug loading.

The term "platelet" refers to cells that are 1-3 microns in diameter, lack a nucleus, but contain RNA. Platelets can contain alpha and dense granules within and contain such factors as P-selectin and serotonin, respectively. Platelets also have an open canalicular system, which refers to channels that are pathways for transport of extracellular substances into the cell and release of substances from the granules into the extracellular environment. They function primarily in the regulation of hemostasis by participating in blood clotting, but have also been shown to have a role in inflammation.

For the purposes of this disclosure, the term "platelets" refers to platelet-like cells, platelet variants or artificial platelets (collectively referred to herein as "platelets") produced according to one or more methods of the present disclosure. PLCs” or “PLC”). In some embodiments, PLCs are derived from iPSCs according to one or more methods of the present disclosure. The PLCs of the present disclosure are variants of naturally occurring platelets or donor platelets. As such, PLCS exhibit structural diversity, structural deviations, or structural differences, and have unique characteristics that can distinguish PLCs from bone marrow-derived donor platelets. The ability of PLC to differ from donor platelets while retaining many of the functional indicators of naturally occurring platelets makes PLC a readily available surrogate for donor platelets.

The term "donor platelets" refers to bone marrow-derived platelets that are produced physiologically within a mammalian (eg, human) body.

The term "pre-platelets" refers to cells that are 3-10 microns in diameter and do not have a nucleus but do have RNA. Pre-platelets are otherwise morphologically and ultrastructurally similar to platelets, breaking apart by cytoskeletal rearrangements and exiting the intermediate cell stage produced by megakaryocytes to form individual platelets. Configure.

The term "preplatelets" refers to cytosolic extensions from or just released from megakaryocytes. Platelet progenitors break apart by cytoskeletal rearrangement to form individual pre-platelets and platelets.

The term "pluripotent stem cells" refers to embryonic, embryo-derived and induced pluripotent stem cells, regardless of how the pluripotent stem cells are derived, as well as cells derived from all three germ layers of the body. including other stem cells that have the ability to Pluripotent stem cells are functionally defined as stem cells that can have one or more of the following characteristics: (a) can induce teratomas when transplanted into immunodeficient (SCID) mice; (b) capable of differentiating into all three germ layer cell types (e.g., capable of differentiating into ectodermal, mesodermal, and endodermal cell types); or (c) expresses one or more markers of embryonic stem cells (e.g., Oct4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, SSEA-5 surface antigen, Nanog, TRA-1-60 , TRA-1-81, SOX2, REX1, etc.).

The term "induced pluripotent stem cells" (iPS cells or iPSCs) refers to a type of pluripotent stem cells generated by reprogramming somatic cells by expressing a combination of reprogramming factors. iPSCs can be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. Factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, the combination of Oct4 (sometimes referred to as Oct3/4), Sox2, c-Myc, and Klf4. In other embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, the combination of Oct4, Sox2, Nanog, and Lin28. In certain embodiments, at least 2, 3, or 4 reprogramming factors are expressed in the somatic cell to reprogram the somatic cell.

As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment,” etc. refer to not, but refers to reducing the probability of developing a disorder or condition in a subject at risk or susceptible to it.

By "reduce" is meant a negative change of at least 10%, 25%, 50%, 75%, or 100%.

By "reducing cell death" is meant reducing the tendency or probability of a cell to die. Cell death may be apoptotic, necrotic, or by any other means.

By "reduced level" is meant that the amount of analyte in a sample is less than the amount of analyte in a corresponding control sample.

"Reference" means standard or control conditions.

"Specifically binds" recognizes and binds to a polypeptide of the present disclosure, but does not substantially recognize other molecules in the sample, e.g., a biological sample that naturally contains the polypeptide of the present disclosure. means a compound or antibody that does not bind to

The term "subject" or "patient" refers to an animal that is the subject of treatment, observation, or experimentation. Solely by way of example, a subject can be, but is not limited to, a human or non-human mammal, such as a non-human primate, mouse, cow, equine, canine, sheep, or feline. including, but not limited to, mammals including;

The terms "treat," "treating," "treatment," and the like, as used herein refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be understood that treatment of a disorder or condition does not require complete elimination of the disorder, condition or symptoms associated therewith, although they are not excluded.

The terms "comprises," "comprising," "containing," and "having," etc., have the meanings ascribed to them in United States Patent Law, and include "consisting essentially of or "consisting essentially of" similarly has the meaning given in United States patent law. and the term is open-ended, unless the basic or novel features being described do not change due to the presence of more than those described There may be more, but prior art embodiments are excluded.

As used herein, the term "or" is understood to be inclusive, unless otherwise specified or clear from context. As used herein, unless specified otherwise or clear from context, the terms “a,” “an,” and “the” refer to the singular or plural.

Unless otherwise specified or clear from the context, the term "about," as used herein, is within the usual tolerance in the art, e.g., within 2 standard deviations of the mean. You will understand when you enter. About is within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, within 0.5% of a stated value. It can be understood as within 5%, within 0.1%, within 0.05%, or within 0.01%. All numerical values set forth herein are modified by the term about, unless the context clearly dictates otherwise.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions that the variable is a single group or combination of listed groups. The recitation of an embodiment herein for a variable or aspect includes the definition that that embodiment is a single embodiment or in combination with any other embodiment or portion thereof.

Any composition or method provided herein can be combined with any one or more of the other compositions and methods provided herein.

The present disclosure produces megakaryocyte progenitor cells (preMK), megakaryocytes (MK), platelet precursors, preplatelets or platelets from stem cells, e.g., pluripotent stem cells, e.g., clinical grade human induced pluripotent stem cells. are directed to compositions and methods for The method allows continuous production of preMK, MK, platelet precursors, preplatelets or platelets from hematopoietic endothelial cells. The preMKs, MKs, proplatelets, preplatelets and platelets obtained by this method are characterized by: their size range, ploidy profile, biomarker expression, gene expression, granular composition, and growth factors, cytokines and chemokines. It can be distinguished by one or more of the compositions or combinations thereof. The present disclosure is further directed to compositions of such preMKs, MKs, proplatelets, preplatelets and platelets and methods of use thereof for drug delivery.

The presence of secretory granules that naturally enclose multiple proteins that promote clot formation (clotting factors) and tissue repair (cytokines, chemokines, and growth factors) is unique to megakaryocytes and platelets. Exocytosis of megakaryocytes and platelet granules plays an important role in thrombosis, modulation of the immune system, and tissue regeneration. Upon contact-activation at sites of bone marrow injury or vascular injury, megakaryocytes and platelets can selectively release the contents of their secretory granules to initiate local therapeutic responses. Platelets are also expected to accumulate naturally at sites of cancer, where they preferentially adhere to tumors (wounds that never heal), hiding them from the immune system, leading to angiogenesis and tumor metastasis. through their granules that contribute to pro-angiogenic factors such as VEGF and anti-inflammatory cytokines such as TGF-β.

Megakaryocytes and platelets are designed to express molecules (e.g., within their granules) to produce "designer cells" that can be specifically adapted for the expression of clotting factors, cytokines, chemokines, growth factors, and drugs. can be loaded or genetically engineered. These modified cells are more or less sensitive and responsive to agonists and improve or improve clotting time under conditions that normally cause cellular dysfunction or reduce clotting. It can be operated to suppress. Similarly, molecules may be conjugated directly on their surface or packaged in secretory granules, which can target tissues by improving cell specificity and can be brought to therapeutic targets to improve their specificity. In some embodiments, platelet membrane-coated nanoparticles may be used instead of whole platelets.

The present disclosure provides for the production of large numbers of megakaryocyte progenitor cells, megakaryocytes, proplatelets, preplatelets, or platelets under cGMP conditions for clinical use, as well as the targeted and selective release of therapeutic targets. To this end, methods and systems are provided for expressing and/or loading drugs in these cell types.

Aspects of the present disclosure relate to scalable cGMP-compliant stem cell-based processes that enable rapid generation of functional megakaryocytes and platelets. In some aspects of the present disclosure, cGMP-compliant human PSC lines can be engineered to conditionally express specific drugs at the megakaryocyte (immediate cell precursor) or platelet level. For example, megakaryocytes and platelets produced according to the processes of the present disclosure may be directed to package drugs into secretory granules as part of normal megakaryocyte or platelet production. As a result, the resulting "designer" modified megakaryocytes and platelets can contain desired drugs, which can be delivered to targeted sites of injury or disease via normal circulation, thereby , avoiding direct systemic exposure of these factors to the body by intravascular transfusion, reducing the non-specific risk of microaggregation/clotting and immunogenicity.

Aspects of the present disclosure are directed to compositions comprising the iPSC-derived megakaryocyte progenitor cells, megakaryocytes, platelet precursors, pre-platelets or platelets of the present disclosure as a vehicle for drug delivery.

According to some aspects of the present disclosure, methods are disclosed for producing the present megakaryocyte progenitor cells, megakaryocytes, platelet precursors, pre-platelets or platelets from pluripotent stem cells, such as clinical grade hiPSCs. be done. These methods allow continuous production of megakaryocyte progenitor cells from hematopoietic endothelial cells over extended timeframes of up to a week or longer, which can then be differentiated into mature megakaryocytes. These iPSC-derived megakaryocytes can be distinguished by their size range, ploidy profile, biomarker expression, and growth factor, cytokine and chemokine composition or combinations thereof.

In some embodiments, MKs and platelets include, but are not limited to, embryonic stem cells (ESC) (e.g., human embryonic stem cells) and induced pluripotent stem cells (iPSCs) (e.g., human induced pluripotent stem cells) can be derived from pluripotent stem cells. ESCs are pluripotent stem cells derived from the inner cell mass of early pre-implantation embryos called blastocysts. iPSCs are a type of pluripotent stem cells that can be generated from adult cells by inducing timed expression of specific transcription factors. iPSCs can be expanded indefinitely in culture, maintained, and engineered to produce MKs and platelets.

In some embodiments, MKs and platelets also include, but are not limited to, CD34 ⁺ cord blood stem cells (UCB cells) (e.g., human CD34 ⁺ cord blood stem cells), CD34 + mobilized peripheral blood cells (MPB cells) (e.g., CD34 ⁺ mobilized human peripheral blood), or hematopoietic stem cells such as CD34+ bone marrow cells. UCB cells are multipotent stem cells derived from the blood remaining in the placenta and attached umbilical cord after birth. MPB cells are multipotent stem cells derived from volunteers whose stem cells have been mobilized into the bloodstream by administration of G-CSF or similar agents.

In some embodiments, MKs and platelets are mesenchymal stem cells (MSCs) such as, but not limited to, adipose-derived mesenchymal stem cells (AdMSCs) or mesenchymal hepatocytes from other sources. can be derived from other stem cell types such as

AdMSCs are elicited from white adipose tissue, which is elicited from the mesoderm during embryogenesis, is present in all mammalian species, and is located throughout the body. ASCs can serve a wide variety of applications due to their wide availability and ability to differentiate into other tissue types of mesoderm, including bone, cartilage, muscle, and fat.

In the present disclosure, stem cell cultures can be maintained independently of embryonic fibroblast feeder cells and/or animal serum. The method may, in some embodiments, utilize a serum-free, feeder cell-free alternative.

The present disclosure provides methods for producing megakaryocyte progenitor cells (preMK) and megakaryocytes (MK) from stem cells.

In some embodiments, the present disclosure provides a method for megakaryocyte production, comprising expanding pluripotent stem cells under agitation under low-adhesive or non-adherent conditions, comprising: allowing the pluripotent stem cells to form self-aggregating spheroids; differentiating the pluripotent cells into hematopoietic endothelial cells in a first culture medium; A method is provided comprising differentiating into a megakaryocyte progenitor cell. The step of differentiating the pluripotent cells into hematopoietic endothelial cells can be performed on the matrix under adherent conditions. In some embodiments, differentiating the pluripotent cells into hematopoietic endothelial cells is performed under low-adherence or non-adherence conditions to allow the hematopoietic endothelial cells to self-aggregate.

In some embodiments, the present disclosure provides a method for megakaryocyte production comprising differentiating pluripotent cells into hematopoietic endothelial cells in a first culture medium; wherein at least one of the step of differentiating the hematopoietic endothelial cells into megakaryocyte progenitor cells and the step of differentiating the pluripotent cells and the step of differentiating the hematopoietic endothelial cells comprises a matrix-coated three-dimensional structure; Provide a method, which is done above. A three-dimensional structure can be a microcarrier or a microcarrier.

In some embodiments, the present disclosure provides a method for megakaryocyte production comprising differentiating pluripotent cells into hematopoietic endothelial cells in a first culture medium; wherein at least one of differentiating the hematopoietic endothelial cells into megakaryocyte progenitor cells comprises differentiating the pluripotent cells and differentiating the hematopoietic endothelial cells so that the cells are capable of self-aggregation; Methods are provided that are performed under low-adhesion or non-adhesion conditions.

The disclosure further provides methods for producing platelets from MKs.

Production Methods FIG. 1 shows an overall schematic for scalable differentiation of megakaryocyte progenitor cells (preMK), megakaryocytes (MK) and platelets (PLC) from one or more pluripotent stem cells. Of note, however, although the process has been described in the context of pluripotent stem cells, in various embodiments the pluripotent stem cells may be replaced with other types of stem cells, Or it may be supplemented. In some embodiments, two-dimensional (2D) culture is used to generate progenitors, megakaryocytes, and platelets, as shown in FIG. Other embodiments disclosed herein describe a process that utilizes three-dimensional culture to produce desired cells, as shown in FIG.

Stage 0: Preparing for Expansion and Differentiation of Human Induced Pluripotent Stem Cells Matrix-dependent Expansion Culture For matrix-dependent expansion culture, clinical grade pluripotent stem cells (PSCs) are grown in pluripotent stem cell culture medium for supportive use. They can be expanded as colonies by culturing them on matrices without feeder cells. Supportive matrices may be two-dimensional surfaces or three-dimensional structures that allow attachment of cells. In some embodiments, clinical grade human induced pluripotent stem cells may be human induced pluripotent stem cells (iPSCs), but other types of pluripotent stem cells, such as embryonic stem cells, or other Stem cells can also be used.

In some embodiments, the supporting matrix is, as non-limiting examples, tissue culture treated plastic, recombinant vitronectin, recombinant laminin, Matrigel, Geltrex, or any combination of the foregoing. good. In some embodiments, the pluripotent stem cell culture medium is, for example, but not limited to, Essential 8 medium (ThermoFisher), StemFlex medium (Thermofisher), NutriStem medium (Biological Industries), or Other media capable of supporting the maintenance and growth of pluripotent cells are also possible. In some embodiments, cells can be cultured until they reach confluency. In some embodiments, cells can be cultured until they reach a % confluency of 30% to 90%. In some embodiments, the cells are cultured to reach up to 60%, up to 65%, up to 70%, up to 75% confluency. For example, cells are cultured until they reach about 70% confluency. Cells are harvested upon reaching a predetermined maximum percent confluency. In some embodiments, cells can be collected as clumps by dissociation using 0.1 mM to 5 mM EDTA or similar chelators or reagents. For example, cells can be harvested using about 0.5 mM EDTA. In some embodiments, cells can be harvested as single cells by dissociation with, for example, proteolytic enzymes, collagenolytic enzymes, or a combination thereof. For example, cells can be harvested as single cells by dissociation with, eg, recombinant trypsin, such as TrypLE™, or Accutase™. For PSC maintenance/expansion, harvested cells can be resuspended in pluripotent stem cell culture medium.

Highly efficient single-cell passaging techniques can be used to support scale-up expansion of undifferentiated hiPSC cultures. The same methodology is also contemplated for cell banking and scale-up hiPSC seed trains leading to pre-MK production. This approach provides rapid expansion for overall manufacturing capacity, undifferentiated pluripotent cultures with the ability to produce pre-MKs, and yields in systems compatible with cGMP manufacturing and clinical registration. and uniformity of culture performance. Briefly, single-cell iPSC suspensions were prepared using one or more cell dissociation enzymes (such as TrypLE (Thermo Fisher)) followed by feeder-free culture at defined densities. Produced by plating in culture medium, such as NutriStem hPSC XF (Biological Industries). In some embodiments, the culture medium is further supplemented with, for example, a ROCK inhibitor and one or more growth factors. In some embodiments, cultures are plated at densities between 5×10 ³ cells/cm ² and 5×10 ⁴ cells/cm ² with culture intervals of 3 or 4 days. For example, in some embodiments, the culture is about 5×10 ³ cells/cm ² , about 1×10 ⁴ cells/cm ² , about 2×10 ⁴ cells/cm ² , plated at a density of about 3×10 ⁴ cells/cm ² , about 4×10 ⁴ cells/cm ² , or about 5×10 ⁴ cells/cm ² with culture intervals of 3 or 4 days , or at a density of 2×10 ⁴ cells/cm ² at a culture interval of 3 days, hi some embodiments, the cultures are plated at a density of about 1×10 ⁴ cells/cm ^{2 .} , are plated at 4 day culture intervals, or plated at 3 day culture intervals at a density of about 2 x ¹⁰⁴ cells/ ^cm2 . The cell attachment of the cells can be mediated by human serum, hi some embodiments, the cultures may be fed feeder-free culture medium without supplementation 18-22 hours after plating. can be passaged at intervals of 3 or 4 days to achieve predictable and consistent yields over multiple passages.

Matrix-independent 3D expansion cultures Figures 3 and 4 present schematics of directed differentiation of preMK cells using 3D matrices. For matrix-independent 3D expansion culture, clinical grade PSCs can be expanded as self-aggregating spheroids. In some embodiments, this can be achieved by seeding single cells at a density of about 100,000 to about 1.5 million per ml. For example, in some embodiments, single cells can be seeded at 0.5 million per ml.

Cells can be subjected to continuous agitation by slow agitation or gentle shaking in low-adherent or non-adherent conditions in pluripotent stem cell culture media. In some embodiments, feeder-free, serum-free media can be used. Pluripotent stem cell culture media include, but are not limited to, Essential 8 medium (ThermoFisher), StemFlex medium (ThermoFisher), NutriStem medium (Biological Industries), or any other medium known in the art for the maintenance and Other similar media capable of supporting growth are also possible. In some embodiments, the culture medium may be supplemented with a ROCK inhibitor (eg H1152). In some embodiments, the medium may contain an epidermal growth factor family member, eg, heregulin-beta-1. In some embodiments, heregulin-beta-1 medium is used for less than 24 hours (eg, 18-22 hours). In some embodiments, the PSC spheroids reach an overall cell density of about 3 million to about 10 million cells/ml and/or achieve a median spheroid size of about 150 to about 350 μm. up to approximately 5-7 days. In some embodiments, PSC spheroids are cultured until the overall cell density reaches 5 million cells/ml. In some embodiments, PSC spheroids are cultured until cells achieve a median spheroid size of about 250 μm. The culturing step may continue for 3, 4, 5, 6, 7, or 8 days. Where applicable, PSCs can be harvested as single cells by dissociation with proteolytic enzymes, collagenolytic enzymes, or a combination thereof. For example, cells may be separated into single cells by dissociation with, but not limited to, trypsin, recombinant trypsin, such as TrypLE™, Accutase™, or similar reagents known in the art. can be recovered as In some embodiments, single cells are used to initiate another 3D expansion and/or directed differentiation culture.

Preparation for Differentiation In some embodiments, to prepare for differentiation, PSC aggregates are removed from matrix-independent 3D cultures by partially dissociating PSC colonies from matrix-dependent 2D cultures. It can be generated by partially dissociating spheroids or by self-aggregation of single PSCs generated by any method known in the art. In some embodiments, these aggregates are placed in a pluripotent stem cell culture medium, such as, but not limited to, Essential 8 medium (ThermoFisher), StemFlex medium (ThermoFisher), or NutriStem, prior to initiating differentiation. It can be produced in culture media (Biological Industries). In some embodiments, the medium may contain a ROCK inhibitor, such as, but not limited to, Y27632, H1152, or combinations thereof. In some embodiments, the medium may contain soluble laminin, eg, recombinant laminin-521. In some embodiments, the medium may contain an epidermal growth factor family member, eg, heregulin-beta-1. In some embodiments, heregulin-beta-1 medium is used for less than 24 hours (eg, 18-22 hours). In some embodiments, cells can be cultured at 37° C., 5% CO ₂ , 20% O ₂ for 0-72 hours before initiating differentiation.

For matrix-dependent differentiation cultures, aggregates can be attached to a surface. In some embodiments, the deposition step can proceed for about 24 hours, although any time between 1 hour and 24 hours or longer may be used. In some embodiments, the surface may be pre-coated with collagen, laminin, or any other extracellular matrix protein. In some embodiments, human collagen IV can be used to coat the surface. In some embodiments, the matrix-coated surface may be 2D (eg, the bottom of a plastic dish or flask). In some embodiments, the matrix-coated surface may be 3D (eg, smooth or textured spherical microcarriers, or macrocarriers, eg, Raschig rings). Cells on the 3D matrix-coated surface can then be cultured with or without continuous movement. For example, cells can be cultured in roller bottles, spinner flasks, stirred tank bioreactors, vertical wheel bioreactors, packed bed bioreactors, or fluidized bed systems under ultra-low attachment static conditions.

For matrix-independent differentiation cultures, aggregates are grown in low-adherence vessels such as, but not limited to, plates or flasks on orbital shakers, spinner flasks, roller bottles, vertical wheel bioreactors, or stirred tank bioreactors. ) in which it can be subjected to continuous motion by slow agitation or gentle shaking. Cells can transition to stage 1 of differentiation after 0 to 72 hours, eg, about 24 hours.

Stage 1. Generation of hematopoietic endothelial cells In stage 1, the prepared PSC aggregates can be differentiated into hematopoietic endothelial cells. Briefly, some or all of the pluripotent stem cell culture medium is removed and replaced with stage 1 differentiation medium. In some embodiments, the stage 1 differentiation medium is a basal medium, e.g. or animal component-free media (ACF), including StemSpan™-ACF (STEMCELL Technologies, Catalog No. 09855), supplemented with multiple growth factors. In some embodiments, the basal medium contains between 1 and 200 ng/ml each of BMP4 (eg, 50 ng/ml), bFGF (eg, 50 ng/ml), and VEGF (eg, 50 ng/ml). is supplemented with one or more of In some embodiments, the stage 1 medium may be further supplemented with a WNT agonist (eg, CHIR98014 or CHIR99021), laminin (eg, recombinant laminin 521), or any combination thereof. In some embodiments, cells are subjected to hypoxic conditions (e.g., 37°C, 5% _CO2 , 5% _O2 ) for between 2 and 6 days, followed by normoxia (37°C, 5% CO2). ₂ , 20% O ₂ ) for 2 to 6 days. In some embodiments, BMP4 may be added for the first 6-48 hours (eg, 24 hours) and not for the remainder of Stage 1 (FIG. 28). In some embodiments, VEGF and bFGF may be absent for the first 6-48 hours (eg, 24 hours) of Stage 1 and added thereafter (FIG. 28). In some embodiments, daily complete medium changes may be performed throughout Stage 1 by removing spent medium and replacing with fresh Stage 1 medium. In some embodiments, a partial medium change may be performed by removing 10-99% of the spent medium and replacing it with an equal volume of fresh Stage 1 medium. In some embodiments, an additional volume of fresh medium may be added, with the net effect of increasing the total volume of the culture. In some embodiments, instead of replacing or adding fresh Stage 1 medium, specific medium components are spiked into the culture.

In 2D matrix-dependent culture, colony morphology changes to scattered elongated cell clumps by day 2 (FIG. 15). By days 5-6, a confluent adherent layer of hematopoietic endothelial cells is observed, with several three-dimensional structures within the adherent cell layer (Fig. 2, Fig. 15). In matrix-independent 3D culture, stage 1 can proceed in matrix-independent 3D culture, in which self-aggregated spheroids are placed in low-adherence vessels, such as, but not limited to, plates on an orbital shaker or It can be subjected to continuous motion by slow agitation or gentle shaking in a flask, spinner flask, roller bottle, vertical wheel bioreactor, or stirred tank bioreactor. In matrix-independent 3D culture, spheroids grow larger, denser and more uneven as stage 1 progresses (Fig. 15). Approximately 6 days after initiation of stage 1 differentiation, differentiation to hematopoietic endothelium is complete. In some embodiments, differentiation can be considered complete when a confluent adherent layer of hematopoietic endothelial cells is observed, with some three-dimensional structures within the adherent cell layer. In some embodiments, differentiation can be assessed by expression of hematopoietic endothelial markers, such as CD31 and CD34. In some embodiments, hematopoietic endothelial cells may also express CD309 and CD144, or CD309, CD144, CD140a and CD235a.

Stage 2. Generation of depleted megakaryocyte progenitor cells (preMK) from hematopoietic endothelial cells In some embodiments, initiation of megakaryocyte progenitor cell (stage 2) differentiation can occur 4 to 8 days after stage 1 . Briefly, some or all of the stage 1 medium is removed and replaced with 1 volume of stage 2 medium, such as STEMdiff™ APEL™ 2 basal medium (STEMCELL Technologies, Catalog No. 05275). . Such stage 2 medium contains 1 to 200 ng/ml of stem cell factor (SCF) (e.g., at 25 ng/ml), thrombopoietin (TPO) (e.g., at 25 ng/ml), Fms-related tyrosine kinase 3 ligand ( Flt3-L) (eg, at 25 ng/ml), interleukin-3 (IL-3) (eg, at 10 ng/ml), interleukin-6 (IL-6) (eg, at 10 ng/ml), and One or more each of heparin (eg, at 5 units/ml) may be supplemented. In some embodiments, the Stage 2 medium may be further supplemented with UM171, UM729, SR-1, SU6656, laminin (eg, recombinant laminin 521), or any combination thereof.

Cells are then incubated at 37° C., 5% CO ₂ , 20% O ₂ for at least 3 days up to 12 days or longer. For example, cells can be incubated for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 days. In some embodiments, a daily partial medium change can be performed by removing 10-99% of the spent medium and replacing it with an equal volume of fresh Stage 2 medium. In some embodiments, an additional volume of fresh medium may be added, with the net effect of increasing the total volume of the culture. In some embodiments, instead of replacing or adding fresh Stage 1 medium, the culture can be spiked with specific medium components.

Within 1-2 days after the onset of stage 2, small round refractile cells appear within the adherent hematopoietic endothelial cells and are eventually released into the supernatant (Fig. 17A, Fig. 24A). These released cells may contain preMKs defined by cell surface expression of CD43 and CD41 and lack of expression of CD14 (FIGS. 17B, 17C, 24B). In 2D culture, floating and weakly attached stage 2 cells that appear on top of the adherent cell layer can be harvested by collecting the medium by gentle gentle rinsing periodically. In 3D culture, released stage 2 cells can be collected (eg, by discontinuing agitation) in a manner that allows aggregates to settle to the bottom of the vessel. Media associated with the released suspended cells can then be collected. Other exemplary methods for collecting released cells can include, for example, exploiting the size and density differences between aggregates and released single cells. Half medium replacement can then be initiated by adding half the original volume of fresh Stage 2 medium on top of the rinsed adherent cell layer or 3D aggregates. In some embodiments, a ratio other than half is used for medium exchange. An aliquot of cells collected in medium can be removed for viable cell enumeration and biomarker analysis by flow cytometry. The remainder of the cells can then be concentrated by centrifugation, counter-centrifugal elutriation, acoustic separation, or any other relevant technique. After concentration, medium replacement can be completed by adding back half the original volume of conditioned medium to the adherent cell layer or 3D aggregates. In some embodiments, an additional volume of fresh medium may be added, with the net effect of increasing the total volume of the culture. In some embodiments, instead of replacing or adding fresh Stage 2 medium, the culture may be spiked with specific medium components. The rest of the supernatant is discarded and the cell pellet containing preMK can be stored at −180° C. in Cryostor 10 cryopreservation medium or transferred directly to Stage 3. In some embodiments, cells containing preMK may be harvested over a period of 2 to 7 days (e.g., 3 days) and additionally cultured in stage 2 medium or other medium in separate containers. You may Once the final harvest is complete, the preMK-containing cells may be pooled together and stored at −180° C. in Cryostor 10 cryopreservation medium (eg Cryostor 10) or transferred directly to Stage 3.

Stage 3. Generation of Mature Megakaryocytes (MKs) from Megakaryocyte Progenitor Cells In some embodiments, differentiation of mature megakaryocytes can be initiated using PSC-derived preMKs generated as described above. Fresh or thawed megakaryocyte progenitor cells can be seeded onto non-adherent surfaces, eg, in Stage 3 medium containing StemSpan™-ACF. A non-adherent surface refers to a surface intended for the majority of cells not to stick or stick to such surface, but instead remain predominantly in suspension. For example, such surfaces may be made of "ultra-low tack plastic" or may not be coated with extracellular matrix proteins to prevent or minimize cell adhesion to the surface. . In some embodiments, the stage 3 medium comprises TPO (eg, 25 ng/ml), SCF (eg, 25 ng/ml), IL-6 (eg, 10 ng/ml), each from 0 to 200 ng/ml. One or more of IL-9 (eg, 10 ng/ml), heparin (eg, 5 units/ml), and a ROCK inhibitor (eg, Y27632 at 5 μM) may be supplemented. In some embodiments, stage 3 medium may also be supplemented with UM171, UM729, SR-1, SU6656, or any combination thereof.

Cells are then incubated at between 37° C. and 40° C. (eg, 39° C.) with between 5 and 20% CO ₂ (eg, between 7% and 10%) and between 5 and 20% O ₂ . It can be incubated for up to 5 days. In some embodiments, daily partial medium changes are performed by removing 10-95% of the spent medium and replacing it with an equal volume of fresh Stage 3 medium. In some embodiments, the non-adhesive surface is an ultra-low adhesion plate or flask. In some embodiments, the non-adhesive surface is a gas permeable membrane (eg, G-Rex®). In some embodiments, the non-adhesive surface is a cell culture bag or vessel with gentle agitation. In both cases, preMKs (either freshly harvested from stage 2 cultures or thawed from cryopreserved stocks) were suspended in stage 3 medium at a density of 100,000-10 million per ml, introduced into the container. For example, preMK is 1-1.5 million per ml, 1-2 million per ml, 1-3 million per ml, 1-4 million per ml, 2-5 million per ml, at a density of 2 to 6 million per ml, 3 to 7 million per ml, 3 to 8 million per ml, 5 to 9 million per ml, or 8 to 10 million per ml good too. In some embodiments, preMK is 100,000 to 1.5 million per ml, 200,000 to 1.5 million per ml, 300,000 to 1.5 million per ml, 0.400,000 to 1.5 million per ml, 0.5 million per ml to 1.5 million, 600,000 to 1.5 million per ml, 700,000 to 1.5 million per ml, 800,000 to 1.5 million per ml, or 900,000 to 1.5 million per ml. Cells are cultured for a total of 1-5 days (eg, 3 days) to allow differentiation into mature MKs. In some embodiments, daily half-medium changes are performed by removing 10-95% of the spent medium and replacing it with an equal volume of fresh Stage 3 medium. At the end of stage 3 culture, the resulting cells have increased in size and ploidy and display many features indicative of mature megakaryocytes (e.g., shown in Figures 34 to 42 and as described below). .

In some embodiments, during Stage 3, megakaryocyte progenitor cells differentiate into mature MKs within a few days. In some embodiments, the initial uniform, small, round, refractile cells (Figure 20A) begin to increase in size and ploidy on days 2-4 (Figures 20B, 20C). At the same time, MKs producing platelet precursors can be readily observed (Fig. 20D). At days 3-4 of stage 3, the proportion of CD61 ⁺ (megakaryocyte lineage) cells co-expressing the mature MK markers CD42a and CD42b increases dramatically and can reach levels above 90% (FIG. 21B). . In some embodiments, mature MKs in stage 3 cultures are stimulated by adjusting the concentrations of heparin and ROCK inhibitors over the last 1-24 hours (e.g., 5 hours) of stage 3. can be induced to produce This effect can be used as a readout of the ability to form platelet precursors and/or to synchronize and augment platelet production in the environment of a platelet bioreactor or other system designed for platelet production. can be used. For example, in some embodiments, stage 3 differentiation is performed in a millifluidic bioreactor, e.g., as described in PCT/US18/21354, the contents of which are incorporated herein by reference in their entirety. .

Stage 4. Platelet and pre-platelet production from mature megakaryocytes After stage 3, platelets are produced in the culture medium by mature MKs. These platelets can be harvested, quantified, and evaluated by flow cytometry, electron microscopy, and fluorescence microscopy, thereby confirming their identity as authentic platelets (FIGS. 44A and 44B). . In some embodiments, platelets are released after 4-5 days.

Platelets and pre-platelets are produced from iPSC-derived megakaryocytes by repeatedly subjecting MKs to shear stress. In some embodiments, this can be achieved by seeding a millifluidics bioreactor with mature megakaryocytes. (FIGS. 63A-66E). Various non-limiting embodiments of suitable bioreactors are described in US9,795,965; US2017/0183616; US2018/0334652; WO2018165308, which are incorporated herein by reference in their entirety.

Additional methods for producing platelets are contemplated herein. For example, platelets can be produced using the method disclosed in US 9,763,984, the contents of which are incorporated herein by reference in their entirety. Briefly, stage 3 megakaryocytes are derived from one or more of: thrombopoietin, or stem cell factor (SCF), thrombopoietin (TPO), interleukin-11, at least one ROCK inhibitor, and heparin. Contact may be made with a hematopoietic cell expansion medium containing at least one selected reagent. In some embodiments, culture media and growth factors similar to those for Stage 3 can be used. Platelets produced by this method may be loaded with a therapeutic agent or genetically modified to contain an agent of interest.

3D-based packed bed bioreactor In some embodiments, a 3D scalable packed bed bioreactor is used for the production of one or more of preMK, megakaryocytes, platelets, or megakaryocytes and platelets can be done. In some embodiments, packed bed bioreactors can be used for stage 1 and stage 2 cultures (Figure 4). For example, packed bed reactors can be used for the differentiation of PSCs into hematopoietic endothelial cells, followed by the production of preMKs. Packed bed reactor carriers can be either micro-sized or macro-sized and can be formed from biocompatible plastics, metals, glass, or natural materials such as alginate. In some embodiments, the carrier is formed from PTFE in the shape of a Raschig ring, eg, a 1 mm Raschig ring. In some embodiments, the carrier may be coated with a matrix as described above. In some embodiments, the carrier may be coated with laminin, eg, laminin 521, a recombinant human protein. In some embodiments, pluripotent cells can be seeded onto the carrier as clumps. In some embodiments, the medium can be removed and replaced with Stage 1 medium at daily medium changes. In some embodiments, during Stage 1, pluripotent cells may exhibit growing regions inside the carriers in the packed bed reactor. In some embodiments, early differentiation of pluripotent cells into hematopoietic endothelium (i.e., stage 1 of directed differentiation), plus further differentiation and release of preMKs (i.e., stage 2 of directed differentiation) can be done in the same container. For example, a packed bed bioreactor may contain laminin-521-coated macrocarriers seeded with pluripotent cells, eg, iPSCs. The packing layer may then be exposed to a continuous flow of medium, which allows stage 1 differentiation into hematopoietic endothelium. After infiltration of the packed bed, the medium can be circulated to a conditioning chamber where fresh medium components are added and oxygen/CO2 concentrations may be adjusted via sparging or other means; The medium can then be recycled to the cells.

Upon completion of stage 1, the medium can be switched to allow stage 2 differentiation and production and release of preMK. Appropriately sized and shaped carriers, such as 1 mm Raschig rings, provide sufficient media flow and channels to allow permeation through the packed bed of released cells and out of the reactor for collection and cryopreservation. width can be enabled. In some embodiments, this design can reduce the shear forces experienced by cells and can allow for efficient media usage due to its perfusion-based design, which when released , preMK.

Self Aggregating Spheroids in Stirred Tank Bioreactors In some embodiments, certain process steps may be performed using scalable 3D solutions, which are performed in a stirred or shaken vessel. Differentiation can be performed using suspended self-aggregating spheroids (Fig. 3). In some embodiments, such containers are coated with hydrophilic or neutrally charged coatings to keep cells in suspension by inhibiting specific and non-specific cell fixation on the surface. may include a low-adhesion surface or a non-adhesion surface that is surface-coated with Pluripotent cells are dissociated into single cells and placed in a pluripotent stem cell culture medium such as, but not limited to, Essential 8 medium (ThermoFisher), StemFlex medium (ThermoFisher), or NutriStem medium (Biological Industries). Can be resuspended. In some embodiments, the maintenance medium may be supplemented with a ROCK inhibitor, such as, but not limited to Y27632, H1152, or combinations thereof. In some embodiments, the medium may contain an epidermal growth factor family member, eg, heregulin-beta-1. In some embodiments, the medium may contain soluble laminin, eg, recombinant laminin-521. In some embodiments, use of heregulin-beta-1 is restricted to less than 24 hours (eg, 18-22 hours). Pluripotent cells may then be incubated in low-adherent or non-adherent vessels and subjected to agitation at standard culture conditions (eg, 37C, 5% CO2, 20% O2). In some embodiments to provide agitation, the incubation vessel may be placed on an orbital shaker, or a shaker or spinner flask, or using a controlled stirred-tank bioreactor, with constant agitation. good too. Within 24 hours, pluripotent cells can self-aggregate to form spheroids approximately 50-150 um in diameter. When agitation is discontinued, the spheroids are allowed to settle to the bottom of the container.

The medium can then be partially or completely replaced with stage 1 differentiation medium to promote differentiation into hematopoietic endothelium, and agitation is performed under hypoxic conditions (e.g., 37°C, 5% CO2, 5% O2). ) can be resumed with incubation at Partial or complete medium changes can be performed periodically (eg, daily), during which time the spheroids can grow larger and develop characteristic structures and shapes. For example, as shown in FIG. 22A, spheroids are cultured for a total of 6 days (4 days at 37° C., 5% CO, 5% O, followed by 2 days at 37° C., 5% CO, 20% O). can be done. As shown in Figure 29A, on day 6, the spheroids are larger, denser, and have a more irregular surface.

To move to Stage 2, agitation may be discontinued and the spheroids allowed to settle to the bottom of the vessel. The medium can then be partially or completely replaced with stage 2 differentiation medium to facilitate differentiation and subsequent release of suspension cells containing preMK. Periodically thereafter (eg, daily), suspension cells may be harvested and partial medium changes may be performed. Released stage 2 cells can be harvested by discontinuing agitation, allowing aggregates to settle to the bottom of the vessel, and collecting the medium associated with the released suspended cells. A medium half replacement can then be initiated by adding half the original volume of fresh Stage 2 medium on top of the adherent cell layer or 3D aggregates. An aliquot of cells collected in medium can be removed for viable cell enumeration and biomarker analysis by flow cytometry. The remainder of the cells can then be concentrated by centrifugation, countercentrifugal elutriation, sonic separation, or other related techniques. After concentration, half medium replacement can be completed by adding back half the original volume of conditioned medium to the adherent cell layer or 3D aggregates. In some embodiments, an additional volume of fresh medium may be added, with the net effect of increasing the total volume of the culture. In some embodiments, instead of replacing or adding fresh Stage 2 medium, the culture may be spiked with specific medium components. The remainder of the supernatant may be discarded and the cell pellet containing preMK may be pooled or transferred directly to Stage 3. In some embodiments, cells containing preMK may be harvested over a period of 2 to 7 days (e.g., 3 days) and additionally cultured in stage 2 medium or other medium in separate vessels. You may Once the final harvest is complete, the preMK-containing cells may be pooled together and either pooled or transferred directly to Stage 3. In some embodiments, preMKs may be cryopreserved for storage. For example, preMKs may be stored at -180°C in Cryostor 10 cryopreservation medium.

When transferred to Stage 3 static culture, preMKs from 3D self-aggregating spheroid cultures can yield similar MK purity as preMKs from 2D culture systems. Furthermore, stage 3 differentiated cultures generated from 3D self-aggregating spheroid cultures dramatically increased in size, consistent with their bona fide megakaryocyte identity, cells capable of generating platelet precursors. can contain

Stage 5. Drug loading in preMK, MK, preplatelets, and platelets Referring to Figures 5-7, defined cell types (e.g. pre-platelets from stage 4, and platelets from stage 4) can be modified to retain or express therapeutic compositions. In some embodiments, these cell types are harvested for purposes of encapsulation and/or surface conjugation of therapeutic compositions (among other methods). In some embodiments, the therapeutic composition comprises a small molecule, biologic, nucleic acid, or any other form of therapeutic agent, including those described below. A therapeutic agent of interest loaded onto or onto any of the cell types can be used for a variety of indications including (but not limited to) cancer, hematologic disease, liver disease, lung disease. .

In some embodiments, target cells can be loaded with the therapeutic composition by incubating the therapeutic composition with the cell suspension. In some embodiments, therapeutic agents are actively taken up by cells (e.g., receptor-dependent uptake), while in other embodiments, therapeutic agents are passively taken up by cells (e.g., platelet and/or by passive diffusion, eg, receptor-independent endocytosis). In some embodiments, the methods of the invention do not require physical or chemical modification of cells for efficient uptake of therapeutic compositions. A therapeutic agent that is taken up by a cell is stored intracellularly, eg, in secretory granules of the cell.

In some embodiments, therapeutic compositions are conjugated to the surface of cells (ie, megakaryocyte progenitor cells, mature megakaryocytes, pre-platelets, and platelets). In some embodiments, conjugation requires cell surface functionalization using techniques known to those of skill in the art. In some embodiments, the therapeutic composition will comprise a reactive moiety capable of binding to or otherwise interacting with a cell surface or functional groups on the cell surface.

In some embodiments, iPSCs may be genetically engineered to express transgenes for production of therapeutic polynucleotides or polypeptides followed by directed differentiation of iPSCs into megakaryocytes and platelets. . Viral transduction and other methods for transgene delivery to iPSCs result in stable expression of biologics that can be used as drugs with efficient transcription and translation in pre-MKs, MKs, and PLCs can be used for

Megakaryocytes and Platelets In some embodiments, the present disclosure provides megakaryocyte progenitor cells, megakaryocytes, pre-platelets, platelet precursors or platelets derived from PSC cells or cell lines in vitro. According to aspects of the present disclosure, megakaryocyte progenitor cells, megakaryocytes, pre-platelets, platelet precursors or platelets derived from PSC cells or cell lines are described in US Pat. 9,763,984 or using the bioreactor disclosed in International Application No. PCT/US2018/021354.

In some embodiments, the disclosure provides an isolated population of cells comprising megakaryocytes or megakaryocyte progenitor cells.

In some embodiments, the present disclosure provides compositions containing megakaryocytes or megakaryocyte progenitor cells. In some embodiments of the present disclosure, compositions comprising megakaryocytes, megakaryocyte progenitor cells or products thereof are disclosed.

According to some embodiments of the present disclosure, megakaryocytes, megakaryocyte progenitor cells or products thereof are homogeneous in shape, size and/or phenotype. that megakaryocytes, megakaryocyte progenitor cells or products thereof of the present disclosure may contain variability in biomarker expression, size, ploidy, number and purity that is characteristically different from variability in corresponding human cells should be understood. In some embodiments, such variability can be significantly reduced. In some embodiments, cell populations can be created to have a desired variability that is lower or higher than that of naturally occurring cells.

In some embodiments, megakaryocyte progenitor cells (preMK) are characterized by expression of the markers CD43 and CD41, and lack of CD14 (ie, CD14 ⁻ , CD41 ⁺ , CD43 ⁺ ). Additional expression of CD42b may indicate that megakaryocyte progenitor cells are in the process of terminal maturation into mature megakaryocytes. In certain embodiments, megakaryocyte progenitor cells generated under differentiation culture are non-adherent and can float freely in the culture medium.

In some embodiments, the megakaryocyte is one or more of CD42a ⁺ , CD42b ⁺ , CD41 ⁺ , CD61 ⁺ , GPVI ⁺ , and DNA ⁺ . In some embodiments, the megakaryocyte is one or more of CD42a ⁺ , CD42b ⁺ , CD41 ⁺ , CD61 ⁺ , and DNA ⁺ . In some embodiments, the megakaryocyte is one or more of CD42b ⁺ , CD61 ⁺ , and DNA ⁺ . In some embodiments, the megakaryocyte is one or more of CD42a ⁺ , CD61 ⁺ , and DNA ⁺ . In some embodiments, the megakaryocyte is one or more of CD42a ⁺ , CD41 ⁺ , and DNA ⁺ . In some embodiments, the megakaryocyte is one or more of CD42b ⁺ , CD41 ⁺ , CD61 ⁺ , and DNA ⁺ . In some embodiments, the megakaryocyte is one or more of CD42b ⁺ , CD42a ⁺ , CD61 ⁺ , and DNA ⁺ . In some embodiments, the megakaryocyte is one or more of CD42b ⁺ , CD42a ⁺ , CD41 ⁺ , and DNA ⁺ . In some embodiments, the megakaryocyte is CD41 ⁺ CD61 ⁺ CD42b ⁺ GPVI ⁺ . In some embodiments, the megakaryocyte is CD41 ⁺ CD61 ⁺ CD42a ⁺ GPVI ⁺ .

In some embodiments, the megakaryocyte is CD61 ⁺ and DNA ⁺ and is about 10-50 μm in diameter. In some embodiments, the average size of the megakaryocytes produced by the methods described herein is between 10 μm and 20 μm, between 11 μm and 19 μm, between 12 μm and 18 μm, between 13 μm and 17 μm, 14 μm to 16 μm, and between 14 μm and 15 μm. In some embodiments, the average size of megakaryocytes produced by the methods described herein is 14.5 μm. In some embodiments, the megakaryocyte has a diameter of about 10-20 μm. In some embodiments, the megakaryocyte has a diameter of about 10-30 μm. In some embodiments, the megakaryocyte has a diameter of about 10-40 μm. In some embodiments, the megakaryocyte has a diameter of about 10-50 μm. In some embodiments, the megakaryocyte has a diameter of about 20-40 μm. In some embodiments, the megakaryocyte has a diameter of about 25-40 μm.

In some embodiments, the megakaryocytes produced by the methods described herein have a polyploidy of 2N-16N. In some embodiments, the megakaryocyte has a ploidy of at least 4N, 8N, or 16N. In some embodiments, the megakaryocyte has a polyploidy of 4N-16N. In some embodiments, the megakaryocytes produced by the methods described herein have 16% +/−11. 4%.

In some embodiments, at least 50% of the megakaryocyte population produced by the methods described herein are CD61 ⁺ and DNA ⁺ and have a polyploidy of 2N to 16N. For example, megakaryocytes (ie beta-1-tubulin positive stage 3 cells) from a representative iPSC differentiation culture have a size range of about 9 μm to about 27 μm with a median of 15 μm (FIG. 40B). . This average size is comparable to 'normal' megakaryocytes from various bone marrow sources as well (Fig. 40C).

In some embodiments, the isolated population or composition of cells contains at least 50% CD42b ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 55% CD42b ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 65% CD42b ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 60% CD42b ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 70% CD42b ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 75% CD42b ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 80% CD42b ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 85% CD42b ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 90% CD42b ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 95% CD42b ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 98% CD42b ⁺ CD61 ⁺ DNA ⁺ cells.

In some embodiments, the isolated population or composition of cells contains at least 50% CD42b ⁺ CD41 ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 55% CD42b ⁺ CD41 ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 65% CD42b ⁺ CD41 ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 60% CD42b ⁺ CD41 ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 70% CD42b ⁺ CD41 ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 75% CD42b ⁺ CD41 ⁺ CD61 ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 80% CD42b ⁺ CD41 ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 85% CD42b ⁺ CD41 ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 90% CD42b ⁺ CD41 ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 95% CD42b ⁺ CD41 ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 98% CD42b ⁺ CD41 ⁺ CD61 ⁺ DNA ⁺ cells.

In some embodiments, the isolated population or composition of cells contains at least 50% CD42b ⁺ CD42a ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 55% CD42b ⁺ CD42a ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 65% CD42b ⁺ CD42a ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 60% CD42b ⁺ CD42a ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 70% CD42b ⁺ CD42a ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 75% CD42b ⁺ CD42a ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 80% CD42b ⁺ CD42a ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 85% CD42b ⁺ CD42a ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 90% CD42b ⁺ CD42a ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 95% CD42b ⁺ CD42a ⁺ CD61 ⁺ DNA ⁺ cells. In some embodiments, the isolated population or composition of cells contains at least 98% CD42b ⁺ CD41 ⁺ CD61 ⁺ DNA ⁺ cells.

In some embodiments, the isolated population or composition of cells contains at least 50% megakaryocytes with a ploidy of 4N or greater. In some embodiments, at least 50% of the megakaryocytes have a polyploidy of 4N-16N. In some embodiments, at least 60% of the megakaryocytes have a polyploidy of 4N-16N. In some embodiments, at least 70% of the megakaryocytes have a polyploidy of 4N-16N. In some embodiments, at least 80% of the megakaryocytes have a polyploidy of 4N-16N. In some embodiments, at least 90% of the megakaryocytes have a polyploidy of 4N-16N. In some embodiments, the isolated population or composition of cells contains megakaryocytes with an average ploidy of 4N.

In some embodiments, the isolated population or composition of cells contains platelet precursors, pre-platelets or platelets generated from the megakaryocytes of the present disclosure. In some embodiments, the platelet precursors, preplatelets or platelets are CD42b ⁺ CD61 ⁺ DNA ⁻ cells. In some embodiments, megakaryocytes are produced by differentiation of hiPSC cells or cell lines in vitro.

In some embodiments, the megakaryocytes produced by the methods described herein comprise one or more of the following: (a) content of MK granules by immunofluorescence microscopy: PF4 for alpha granules and (b) gene expression data: Oct4-, Nanog-, Sox2-, Zfp42-, Zfpm1+, Nfe2+, Runx1+, Meis1+, Gata1+; (c) low fibrinogen, serotonin; and has LDL/does not have fibrinogen, serotonin and LDL, and (d) can take up fibrinogen, serotonin and LDL when incubated with plasma.

In some embodiments, megakaryocytes produced by the methods described herein have characteristic expression profiles of growth factors, cytokines, chemokines, and related factors (Figure 42). In some embodiments, the present disclosure provides platelet-derived growth factor isoforms PDGF-AA or PDGF-BB, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (FGF- 2) hematopoietic growth factors Flt3L, G-CSF, GM-CSF, interleukins (IL-1RA, IL-8, or IL-16), CXC chemokine family members CXCL1 (GROalpha) or CXCL12 (SDF -1), the TNF superfamily members sCD40L or TRAIL, or the CC chemokine family members CCL5 (RANTES), CCL11 (eotaxin-1), CCL21 (6CKine) or CCL24 (eotaxin-2). Compositions or pharmaceutical compositions comprising the present megakaryocytes are provided. In some embodiments, the present disclosure provides compositions or pharmaceutical compositions comprising the subject megakaryocyte lysates. Such lysates can be prepared by any method known in the art, for example, by disrupting the preMK or MK membrane by viral, enzymatic, or osmotic mechanisms that compromise its integrity. can. The lysate, in some embodiments, may contain additional agents or can be prepared as a different composition (liquid, paste, etc.), depending on the needs of a particular application. In some embodiments, such compositions comprise platelet-derived growth factor isoforms PDGF-AA or PDGF-BB, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2), hematopoietic growth factor Flt3L, G-CSF, GM-CSF, interleukin (IL-1RA, IL-8, or IL-16), CXC chemokine family member CXCL1 (GROalpha) or Factors such as CXCL12 (SDF-1), TNF superfamily members sCD40L or TRAIL, or CC chemokine family members CCL5 (RANTES), CCL11 (eotaxin-1), CCL21 (6CKine) or CCL24 (eotaxin-2) can include

In some embodiments, the iPSC-derived platelets are 2-5 μm in diameter (eg, 3 μm in diameter) as determined by flow cytometry using calibration beads of known size in this example, Platelets are larger than 5 μm in diameter (FIG. 43A). These platelets stained negatively for DRAQ, a DNA intercalating dye that detects genomic material, and positively for CD61, a platelet-specific surface receptor, with varying degrees of viable cells within the DRAQ-CD61+ gate ( have been observed to have calcein AM) and CD42a expression. DRAQ-CD61+ cells from human iPSC-derived platelets also stained negatively for CD62p, a marker of platelet activation, suggesting that they were in a properly resting, quiescent state when harvested from culture. (Fig. 45). Several times, PLCs differentiated from iPSC-derived megakaryocytes lack GPVI, a receptor that binds collagen, when assessed against human donor platelets by antibody staining and subsequent flow cytometry (Fig. 46C). ing.

The human iPSC-derived platelets described herein can be distinguished from primary human donor-derived platelets for their lack of GPVI expression and more thrombin generation over a shorter time frame (FIGS. 46C-47). They behaved similarly to human donor-derived platelets in a mouse in vivo laser injury model of thrombus formation in the arterioles of the cremaster, in which, following infusion to a proximal site in the injured blood vessel, they were described herein. Both donor platelets (Fig. 48A) and human iPSC-derived platelets (Fig. 48B) described in the literature are incorporated into the developing thrombus. This data demonstrates a unique feature of human iPSC-derived platelets that do not inhibit their quiescence when harvested from culture, and their ability to contribute to thrombus in vivo (FIG. 48B). These data demonstrate similar readouts of platelet functionality with respect to hemostasis and thrombosis between primary and human iPSC-derived platelets.

In some embodiments, the platelets are one or more of CD61+, DRAQ-, Calcein AM+, CD42a+, and CD62P- (at rest). In some embodiments, the platelets are CD61+, DRAQ-, Calcein AM+, and CD62P-. In some embodiments, the platelets are one or more of CD61+, DRAQ-, Calcein AM+, CD42a+, and CD62P+ (in an activated state). In some embodiments, the platelets are CD61+, DRAQ-, Calcein AM+, and CD62P+. In some embodiments, the platelets differ from donor platelets in that they do not express GPVI on the cell surface, yet CD61+, DRAQ-, calcein AM+, CD42a+, and CD62P- (at rest) and/or one or more of CD61+, DRAQ-, Calcein AM+, CD42a+, and CD62P+ (in an activated state). In some embodiments, the platelets differ from donor platelets in that they do not express GPVI on the cell surface, yet are CD61+, DRAQ-, calcein AM+, CD42a+, CD62P- (in resting state), and /or CD61+, DRAQ-, calcein AM+, CD42a+, and CD62P+ (in activated state). In some embodiments, the platelets differ from donor platelets in that they do not express GPVI on the cell surface, yet are CD61 and CD62P+.

In some embodiments, the platelets have a diameter of about 1 μm. In some embodiments, the platelets have a diameter of about 2 μm. In some embodiments, the platelets have a diameter of about 3 μm. In some embodiments, the platelets have a diameter of about 4 μm. In some embodiments, the platelets have a diameter of about 5 μm. In some embodiments, the proplatelets are 5 μm or larger. In some embodiments, the platelets have a diameter of about 1 μm to about 5 μm. In some embodiments, the platelets have a diameter of about 1 μm to about 4 μm. In some embodiments, the platelets are about 1 μm to about 3 μm in diameter. In some embodiments, platelets are about 3 μm to about 4 μm in diameter. In some embodiments, platelets are about 3 μm to 5 μm in diameter.

In some embodiments, PLCs differentiated from iPSC-derived megakaryocytes are efficient in generating thrombin when stimulated by tissue factor and are observed in platelets from peripheral blood plasma in thrombin generation assays. The lag time is reduced and a higher amount of thrombin is obtained (Figures 47 and 76-78). In some embodiments, thrombin generation in iPSC-derived platelets is 2-3 times greater than in a population of donor-derived platelets. In some embodiments, thrombin generation results in maximal thrombin concentrations of about 250 nM to about 850 nM. In some embodiments, thrombin generation results in maximal thrombin concentrations of about 450 to about 600 nM. In some embodiments, the lag time between stimulation and achieving maximum thrombin concentration in a population of iPSC-derived platelets is reduced compared to the lag time for a population of donor-derived platelets. In some embodiments, the lag time for achieving maximum thrombin concentration in the population of induced pluripotent stem cell-derived platelets is between about 5 minutes and 15 minutes, or between about 7 minutes and 15 minutes, wherein The populations contain approximately 0.5×10 ⁶ platelets/mL to 3×10 ⁶ platelets/mL, respectively. In some embodiments, in a thrombin generation assay (TGA), the maximum PLC-generated thrombin concentration occurs at about 10 minutes. This represents a significant reduction in lag time compared to thrombin generation in donor-derived platelets.

Thrombin generation is quantified by enzymatic conversion of the thrombin substrate to a fluorogenic molecule that can be detected by standard methods. The graph shows rapid generation of thrombin in hiPSC-derived platelet samples, as well as rapid decrease in signal. This effect is in stark contrast to the thrombin generation observed from platelets isolated from peripheral blood over the course of 90 minutes.

In some culture conditions, the number of PLCs used to generate thrombin can affect the maximum thrombin concentration produced and the lag time observed before maximum concentration is reached. . For example, in some embodiments, a culture containing 3×10 ⁶ PLCs produces higher concentrations of thrombin in a shorter period of time compared to a culture containing 0.5×10 ⁶ PLCs. It is expected that In some embodiments, thrombin generation in PLC is 2-3 times greater than thrombin generated in donor-derived platelets. In some embodiments, the maximum thrombin concentration generated by PLC is 250 nM to 850 nM, 250 nM to 700 nM, 250 nM to 650 nM, about 250 nM to 600 nM, about 250 to 550 nM, about 250 to 500 nM, or about 250 nM to 450 nM. . In some embodiments, the maximum thrombin concentration generated by PLC is about 300 nM to 850 nM, about 350 nM to 850 nM, about 400 nM to 850 nM, about 450 nM to 850 nM, about 500 nM to 850 nM, about 550 nM to 850 nM, about 600 nM to 850 nM , about 650 nM to 850 nM, about 700 nM to 850 nM, or about 750 nM to 850 nM. Conversely, donor-derived cells yield maximal thrombin concentrations of less than 200 nM at the same cell density. In some embodiments, the composition may comprise a cell density of about ^0.5x106 to ^3.0x106 to produce the desired thrombin concentration.

In some embodiments, the PLC exhibits certain distinguishing features from those of naturally occurring or donor platelets. These features may indicate unique biological properties or compositions of products produced by iPSC-derived MK bioreactors compared to those harvested from the adult circulatory system. For example, in some embodiments, the distinguishing feature is having a uniform cell age after induction, having a developmental age more similar to embryonic, fetal or neonatal platelets, a specific or altered containing non-platelet particles derived from products that have a similar cell signaling or activation pathway structure or that have a biological function.

In some embodiments, "having a uniform cell age after induction" means that all PLCs produced were induced at the same time. In some embodiments, the production of PLC is carried out in a bioreactor, and thus that the produced cells "have a uniform cell age after induction" means that the newly produced cells (i.e., the same "cell age”, day 0). In contrast, platelets in donor samples are a composite of different cell ages (days 0 to 10). Donor platelets are typically eliminated around day 10. Thus, in some embodiments, the PLC composition wherein at least 60% of the PLCs have the same cell age. In some embodiments, the PLC composition wherein at least 70% of the PLCs have the same cell age. In some embodiments, the PLC composition wherein at least 80% of the PLCs have the same cell age. In some embodiments, the PLC composition wherein at least 90% of the PLCs have the same cell age. In some embodiments, the PLC composition wherein substantially all PLCs have the same cell age.

In some embodiments, the cells produced according to the methods described herein are of a developmental cell age that more closely resembles embryonic, fetal, or neonatal platelets than adult cells. Without being bound by theory, these cells may be of a younger developmental cell age than the adult population.

In some embodiments, cells produced by the methods described herein have specific or altered cell signaling or activation pathway structures. For example, in some embodiments, the PLCs exhibit reduced CD62p production when exposed to TRAP-6 as compared to donor-derived platelets. In some embodiments, the PLC exhibits lack of response to TRAP or thrombin. In some embodiments, the PLC is expected to produce thrombin in TGA without triggering tissue factor signaling (in this assay, donor platelets require activation). In some embodiments, cells containing non-platelet particles derived from biologically functional products such as microvesicles or microparticles are provided. In some embodiments, the non-platelet particles are microvesicles or microparticles.

In some embodiments, the platelets contain secretory granules, an open canalicular system, and a dense duct system (Figure 44A). In some embodiments, the platelets contain a characteristic β1-tubulin ring (FIG. 44B). In some embodiments, the platelets activate on glass surfaces and exhibit filopodia and lamellipodia (FIG. 44B). In some embodiments, the present platelets differ from donor platelets in that they preferentially generate thrombin in a shorter time frame upon stimulation with a procoagulant (Figure 47). In some embodiments, the platelets behave similarly to donor platelets with respect to incorporation into thrombi in injured cremaster arterioles in mice (FIG. 48).

In some aspects, compositions are provided that include platelets that are one or more of CD61+, DRAQ-, Calcein AM+, CD42a+, and CD62P- (at rest). In some embodiments, the platelets are one or more of CD61+, DRAQ-, calcein AM+, CD42a+, and CD62P+ (in activated state). In some embodiments, the composition comprises platelets that do not express GPVI.

Microparticles Another aspect of the present disclosure is a composition comprising a population of induced pluripotent stem cell (iPSC)-derived platelet-derived microparticles or microvesicles, wherein the microparticles are donor-derived platelet-derived microparticles The population of microparticles provides a composition that is derived from approximately the same number of iPSC-derived platelets and donor-derived platelets. In some embodiments, the thrombogenic activity of microparticles derived from induced pluripotent stem iPSC-derived platelets is greater than that present in microparticles derived from donor-derived platelets or megakaryocytes. In some embodiments, the thrombogenic activity present in the microparticles results in a maximum thrombin concentration of about 400 nM. In some embodiments, the average diameter of the microparticles from the iPSC-derived platelet population is less than 50% of the diameter of the microparticles from the donor-derived platelet population having approximately the same number of platelets as the iPSC-derived platelet population. be. In some embodiments, megakaryocytes or platelets are genetically modified to contain a nucleic acid molecule encoding a therapeutic agent.

Microvesicles, or microparticles (used interchangeably herein), are intracellular-sized particles that consist of a membrane lipid bilayer and cellular contents. Platelet-derived microvesicles can exert both anti-inflammatory or pro-inflammatory functions and have potential as vehicles for drug delivery. In some embodiments, the microvesicles are capable of producing thrombin (see Figures 78A and 78B).

In some embodiments, the diameter of the microparticles is 0.1 and 4 μm. In some embodiments, the diameter of the microparticles is 0.1 and 3 μm. In some embodiments, the diameter of the microparticles is 0.1 and 2.5 μm. In some embodiments, the diameter of the microparticles is 0.1 and 2 μm. In some embodiments, the diameter of the microparticles is 0.1 and 1.5 μm. In some embodiments, the diameter of the microparticles is 0.1 and 1.0 μm. In some embodiments, the diameter of the microparticles is 0.1 and 0.9 μm. In some embodiments, the diameter of the microparticles is 0.1 and 0.8 μm. In some embodiments, the diameter of the microparticles is 0.1 and 0.7 μm. In some embodiments, the diameter of the microparticles is 0.1 and 0.6 μm. In some embodiments, the diameter of the microparticles is 0.1 and 0.5 μm. In some embodiments, the diameter of the microparticles is 0.1 and 0.4 μm. In some embodiments, the diameter of the microparticles is 0.1 and 0.3 μm. In some embodiments, the diameter of the microparticles is 0.1 and 0.2 μm. In some embodiments, the diameter of the microparticles is 0.2 and 1 μm. In some embodiments, the diameter of the microparticles is 0.3 and 1 μm. In some embodiments, the diameter of the microparticles is 0.4 and 1 μm. In some embodiments, the microparticles are 0.5 and 1 μm in diameter. In some embodiments, the microparticles are 0.6 and 1 μm in diameter. In some embodiments, the diameter of the microparticles is 0.7 and 1 μm. In some embodiments, the diameter of the microparticles is 0.8 and 1 μm. In some embodiments, the microparticles are 0.9 and 1 μm in diameter. In some embodiments, the diameter of the microparticles is 0.2 and 2 μm. In some embodiments, the diameter of the microparticles is 0.3 and 2 μm. In some embodiments, the diameter of the microparticles is 0.4 and 2 μm. In some embodiments, the microparticles are 0.5 and 2 μm in diameter. In some embodiments, the diameter of the microparticles is 0.6 and 2 μm. In some embodiments, the diameter of the microparticles is 0.7 and 2 μm. In some embodiments, the diameter of the microparticles is 0.8 and 2 μm. In some embodiments, the diameter of the microparticles is 0.9 and 2 μm. In some embodiments, the diameter of the microparticles is 1.0 and 2 μm. In some embodiments, the microparticles are 1.5 and 2 μm in diameter. In some embodiments, the diameter of the microparticles is 2.0 and 2.5 μm.

In some embodiments, microparticulate thrombogenic compositions are provided such that the composition has a peak size of less than approximately 2 μm. In some embodiments, the thrombogenic microparticles range in size from 40 nm to 100 nm in diameter. In some embodiments, the thrombogenic microparticles constitute greater than 50% of the composition. In some embodiments, the thrombogenic microparticles constitute greater than 60%, greater than 70%, greater than 80%, greater than 90% or 100% of the composition.

Megakaryocyte progenitor cells, megakaryocytes, proplatelets, pre-platelets and platelets as drug delivery vehicles Platelets offer the potential to serve as part of a bioavailable drug delivery system. Furthermore, platelets, due to their immunomodulatory and angiogenic functions, are actively recruited by tumors to aid in immune evasion and support their growth and metastasis. According to some aspects of the present disclosure, the ex vivo PSC-derived megakaryocyte progenitor cells, megakaryocytes, platelet precursors, preplatelets, or platelets described herein are used in therapeutic compositions such as drugs, It can be used as a vehicle to deliver small molecules, biologics (eg proteins) or similar therapeutic agents. Benefits of this type of drug delivery include, but are not limited to, the ability to deliver molecules to tissues that are traditionally difficult to target due to limitations imposed by drug permeability or retention; reduction of the need for systemic treatment of the patient by hiding the drug in megakaryocytes/pre-platelets/platelet secretory granules until selective release at therapeutic targets; and unwanted toxicity or immunogenicity avoidance. In some embodiments, this type of drug delivery can also lower the dosage required to achieve the desired therapeutic outcome and reduce systemic toxicity.

The terms "therapeutic composition," "drug," "therapeutic agent," and "agent" are used interchangeably and refer to any small molecule compound, antibody, nucleic acid molecule, polypeptide, or any other biological agent. or refer to fragments thereof. In some embodiments, the therapeutic composition is an agent that binds to the target of interest, an antibody to the target of interest, an agonist or antagonist of the target of interest, a peptidomimetic of the target of interest, It can be a small RNA directed or a mimic of the target of interest, and the like. In some embodiments, therapeutic compositions are capable of modulating the expression and/or activity of a target of interest.

In some embodiments, therapeutic compositions are polypeptides or small molecules. For example, the polypeptide may be atezolizumab, a fully humanized monoclonal antibody. The additional polypeptide may be ipilimumab, bevacizumab, cetuximab, or trastuzumab. Examples of small molecules include, but are not limited to, aripiprazole, esomeprazole, or rosuvastatin.

In some embodiments, the therapeutic composition comprises an anti-angiogenic or chemotherapeutic agent suitable for treating, inhibiting, and/or preventing cancer. Examples of anti-angiogenic agents include, but are not limited to, doxorubicin, a DNA damaging agent. Additional examples include vincristine, irinotecan, and paclitaxel.

In some embodiments, the therapeutic composition comprises a growth factor such as, but not limited to, VWF, keratinocyte growth factor, clotting factor (e.g., FVII, FVIII, FIX), epidermal growth factor, or hair growth factor . FVIIa is an activated coagulation factor that has been shown to be of benefit in patients with uncontrolled bleeding. To achieve this effect, FVIIa is administered systemically at high costly concentrations and has been shown to cause thrombotic complications in some patients. FVIIa-supplemented megakaryocyte progenitor cells, megakaryocytes, pre-platelets, or platelets generated according to the processes of the present disclosure can significantly improve hemostasis and survival in the short term following injury. Factor VIII participates in blood clotting, it is a cofactor for factor IXa, a complex that converts factor X to activated factor Xa in the presence of Ca2+ and phospholipids. form the body. In humans, factor VIII is encoded by the F8 gene. Absence of this gene results in hemophilia A, an X-linked recessive clotting disorder. In response to injury, clotting factor VIII is activated and separated from von Willebrand factor to become FVIIIa. Factor IX is a serine protease in the coagulation system. Deficiency of this protein causes hemophilia B. Factor IX is produced as an inactive precursor, the zymogen. two chains that are processed to remove the signal peptide, glycosylated, and then cleaved by Factor XIa (of the contact pathway) or Factor VIIa (of the tissue factor pathway) to link the chains with disulfide bridges form occurs. When activated to factor IXa in the presence of Ca2+, membrane phospholipids, and a factor VIII cofactor, it hydrolyzes one arginine-isoleucine bond in factor X to release factor Xa. Form.

In some embodiments, the therapeutic composition comprises a chemokine or growth factor, such as platelet-derived growth factor isoforms (PDGF-AA, -AB and -BB), transforming growth factor-b (TGF-b), insulin-like growth factor-1 (IGF-1), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF or FGF-2) , hepatocyte growth factor (HGF), connective tissue growth factor (CTGF), and bone morphogenetic proteins 2, -4 and -6 (BMP-2, -4, -6).

In some embodiments, therapeutic compositions are proteins. In some embodiments, the protein is a cytokine, such as interleukin-1-beta, interleukin-2, or interleukin-12. In some embodiments the protein is an antibody protein. In some embodiments, the antibody is atezolizumab or ipilimumab.

Preplatelets, preplatelets and platelets store bioactive factors in secretory granules that they acquire from megakaryocytes. In some embodiments, the platelet precursors, pre-platelets and platelets of the present disclosure may be modified to store or otherwise retain therapeutic compositions.

In some embodiments, platelets may be engineered to express proteins on their surface, or otherwise tagged with proteins on their surface, or various They may be engineered to express antibodies or molecules, or cultured to "load" various antibodies or molecules into their secretory granules. Such engineered platelets can be used to transport and direct therapeutic compositions to desired tissues (or therapeutic targets). In some embodiments, genetic manipulation can be performed at the PSC level or at the megakaryocyte progenitor cell level to conditionally control expression to , or to be expressed in platelets.

Some aspects of the present disclosure relate to modified megakaryocytes, proplatelets, preplatelets or platelets that express desired characteristics for targeting applications. These tools fulfill the promise of personalized medicine without the drawbacks that have hindered commercial implementation (high cost, time consuming, and inability to scale products). can be used to achieve the desired results. Rather than generating custom hPSC lines from individual donors, the priority is to develop platforms that utilize optimized cGMP-compliant hPSC lines for manufacturing therapeutic products. Genetic controls of hPSC lines can then be applied to generate targeted therapeutics and designer products for recipients.

In some embodiments, the modified megakaryocyte progenitor cells, megakaryocytes, proplatelets, preplatelets, or platelets express a protein (including polypeptides, peptides) of interest. In some embodiments, the engineered megakaryocyte progenitor cells, megakaryocytes, proplatelets, preplatelets, or platelets express high levels of a protein of interest (including a polypeptide or peptide). In some embodiments, the megakaryocyte progenitor cell, megakaryocyte, proplatelet, preplatelet, or platelet reduces or suppresses expression of a protein (including polypeptide or peptide) of interest are genetically modified. In some embodiments, the megakaryocyte progenitor cells, megakaryocytes, platelet precursors, pre-platelets, or platelets are modified to express or overexpress a protein (including a polypeptide or peptide) of interest. is genetically modified to reduce or suppress the expression of a protein (including polypeptide or peptide) of , or any combination of the foregoing. For example, in some embodiments, the modified megakaryocyte progenitor cells, megakaryocytes, platelet precursors, pre-platelets, or platelets are enriched in their granules with high levels of clotting factors such as Factor VIIa, Factor VIII It expresses Factor, Factor IX, or VWF, thereby enhancing clot formation at the site of injury without risking systemic hypercoagulability. In some embodiments, the modified megakaryocyte progenitor cells, megakaryocytes, proplatelets, pre-platelets, or platelets are targeted to trauma and are used in the first "critical time" after severe traumatic injury. increases the effectiveness of platelet transfusions during Another potential application of the subject engineered megakaryocyte progenitor cells, megakaryocytes, preplatelets, or platelets is in the treatment of fetal and neonatal alloimmune thrombocytopenia (FNAIT). In this condition, the maternal immune system targets fetal platelets that express the human platelet antigen (HPA) that the mother does not express, resulting in fetal thrombocytopenia and serious potential complications (fetal cranial thrombocytopenia). (including internal bleeding). In some embodiments, the condition is treated using the subject megakaryocyte progenitor cells, megakaryocytes, proplatelets, preplatelets, or platelets that have been engineered with a single base pair alteration, and do so. HPA (negative) platelets or microparticles are administered to HPA-positive children after delivery by an HPA-negative woman, thereby preventing HPA-associated clearance.

In some embodiments, the modified megakaryocyte progenitor cells, megakaryocytes, platelet precursors, preplatelets, or platelets deliver growth factors. In some embodiments, the modified megakaryocyte progenitor cells, megakaryocytes, proplatelets, preplatelets, or platelets loaded with or expressing growth factors are used in cell culture, tissue regeneration, wound Used for healing, functional cosmetics, and hemostatic bandages.

In some embodiments, the megakaryocyte progenitor cell, megakaryocyte, proplatelet, preplatelet, or platelet is treated with an immune checkpoint inhibitor, such as, but not limited to, anti-PDL1, anti-PD1, anti-VEGF, It has been modified to deliver anti-cancer drugs such as anti-CD20 and anti-CTLA4, or anti-CCR4, or anti-PI3K. In some embodiments, the megakaryocyte progenitor cells, megakaryocytes, platelet precursors, preplatelets, or platelets contain cytokines, such as, but not limited to, interleukin-1 beta, interleukin-2, or interleukin-12. modified to deliver

Loading iPSC-Derived Megakaryocyte Progenitor Cells, Megakaryocytes, Preplatelets, Pre-Platelets or Platelets with Therapeutic Compositions Referring to FIGS. The therapeutic composition can be loaded onto or onto sphere progenitor cells, megakaryocytes, platelet precursors, pre-platelets or platelets.

In some embodiments, the megakaryocyte progenitor cells, megakaryocytes, proplatelets, preplatelets, or platelets can be loaded with a therapeutic composition, as shown in FIG. For example, these cells can be loaded with small molecules by co-incubating them with the cells in an aqueous solution (Figure 5). In some embodiments, the therapeutic composition contains 10 ⁵ to 10 ⁷ cells, such as 10 ⁶ cells, at a concentration of 1-200 μM (eg, 100 μM) in a solution or dialysis cassette. Present with suspended matter. The combination of therapeutic composition and cell suspension is incubated at room temperature or at 37° C. for 30 minutes to 24 hours, eg 4 hours. In some embodiments, loading the cells in the cell suspension with the therapeutic composition is facilitated by constant agitation in a dialysis cassette (i.e., a slide-a-lyzer dialysis device from Thermo Fisher Scientific). , thereby allowing platelets to remain "quiescent" during the loading process.

In some embodiments, therapeutic agent compositions, such as those described above, are applied to iPSC-derived platelets, megakaryocytes, megakaryocyte progenitor cells, and pre-platelets by passive loading (also known as “sponge loading”). can be loaded. In some embodiments, passive loading includes MK, MK progenitor, pre-platelet, and/or platelet cells in an aqueous buffer at room temperature or 37° C. for 1 to 5 hours, such as 2 hours. This is accomplished by adding the therapeutic composition to the suspension. In some embodiments, excess therapeutic agent is washed out of the cell suspension by diluting the cell suspension 2- to 10-fold, such as 5-fold. The diluted cell suspension is then centrifuged, the supernatant removed, and the therapeutic-loaded cell suspension resuspended in fresh medium.

Without being bound by theory, incubating the present megakaryocyte progenitor cells, megakaryocytes, pre-platelets, or platelets with a therapeutic composition may induce therapeutic conversion of the cells to secretory granules (e.g., alpha granules, dense granules). Cause entrapment of the composition. In some embodiments, the therapeutic composition-loaded megakaryocyte progenitor cells, megakaryocytes, pre-platelets, or platelets are used to treat sites of inflammation, vascular injury, tissue regeneration, lymphangiogenesis, It can be used to locally deliver therapeutic compositions to target sites such as cancer development, progression and metastasis.

Referring to FIG. 6, iPSC-derived platelets, megakaryocytes, megakaryocyte progenitors, and pre-platelets also share therapeutic compositions to cell membranes, among other organelles, subcellular compartments, and cellular structures. A therapeutic composition may be loaded by conjugation by binding. Covalent conjugation has been performed using high-performance methods including (but not limited to) thiolation of membrane proteins with sulfhydryl-reactive crosslinkers (Fig. 54), alkyne-reactive azides, biotin and avidin (and avidin analogues). It can be accomplished using a variety of bioconjugation techniques such as affinity binding agents, docking of antibodies to membrane-bound epitopes, and other methods. In some embodiments, covalent conjugation of therapeutic compositions converts amines present in amino acids in therapeutic agents to succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (Fig. 54B) and reacting for 1 to 4 hours, such as 2 hours (Figure 54B). In some embodiments, platelets (and other cell types) are functionalized with reactive thiol groups by conversion from primary amines using 2-iminothiolane, also known as Traut's reagent (Sigma) ( Figure 54A). In some embodiments, a therapeutic agent that has reacted with SMCC is co-incubated with a cell suspension treated with Traut's reagent to complete chemical conjugation of the therapeutic composition to the membrane of the cell. (Fig. 54C).

Genetic Manipulation of iPSC-Derived Megakaryocyte Progenitor Cells, Megakaryocytes, Preplatelets, Pre-Platelets or Platelets to Express Therapeutic Compositions Referring to FIG. Progenitor cells, megakaryocytes, proplatelets, preplatelets or platelets can be engineered to express a protein of interest (including a polypeptide or peptide of interest). Such alterations can be made at the stem cell level or at any other level during the platelet production process of the present disclosure. For example, the present disclosure provides a modified megakaryocyte or megakaryocyte progenitor cell differentiated from an engineered hPSC cell or cell line, wherein the modified megakaryocyte or megakaryocyte progenitor cell contains a protein of interest ( A megakaryocyte or megakaryocyte progenitor cell expressing a polypeptide or peptide) is provided. In some embodiments, the engineered megakaryocyte or megakaryocyte progenitor-derived platelet precursors, pre-platelets or platelets can also express a protein of interest and deliver the protein of interest to a target site. can be used for

In some aspects of the present disclosure, PSC-derived megakaryocytes are engineered to express at least one peptide, polypeptide or protein of interest. In still other aspects of the present disclosure, the PSCs are engineered to express at least one peptide, polypeptide or protein of interest, megakaryocytes expressing at least one peptide, polypeptide or protein of interest, or at least Pre-platelets or platelets containing one peptide, polypeptide or protein of interest can be prepared using the methods of U.S. Patent No. 9,763,984 or the bioreactor disclosed in International Application No. can be produced, which are incorporated herein in their entireties by this reference.

In some aspects of the present disclosure, PSC-derived megakaryocytes are engineered to contain DNA or RNA of interest. In still other aspects of the present disclosure, PSCs are engineered to contain DNA or RNA of interest. In some embodiments, pre-platelets or platelets derived from modified megakaryocytes or megakaryocyte progenitor cells containing the DNA or RNA of interest are capable of delivering the DNA or RNA of interest.

Some embodiments relate to compositions or isolated populations comprising engineered PSCs that have been engineered to express at least one peptide, polypeptide or protein of interest. In some embodiments the protein is a cytokine, chemokine or growth factor. Some embodiments relate to a composition or isolated population comprising megakaryocytes engineered to express at least one peptide, polypeptide or protein of interest. In some embodiments the protein is a cytokine, chemokine or growth factor. Some embodiments relate to compositions comprising platelets produced ex vivo from megakaryocytes engineered to express at least one peptide, polypeptide or protein of interest. In some embodiments the protein is a cytokine, chemokine or growth factor. Pre-platelets or platelets produced by modified megakaryocytes expressing at least one peptide, polypeptide or protein of interest are delivered to deliver the at least one peptide, polypeptide or protein of interest to the site of interest. Can be used as a vehicle. In some embodiments the protein is a cytokine, chemokine or growth factor.

Some embodiments relate to compositions or isolated populations comprising engineered PSCs engineered to contain DNA or RNA of interest. Some embodiments relate to compositions or isolated populations comprising megakaryocytes engineered to contain DNA or RNA of interest. Some embodiments relate to compositions comprising platelets produced ex vivo from megakaryocytes engineered to contain DNA or RNA of interest. Modified megakaryocyte-produced platelet precursors, pre-platelets or platelets containing the DNA or RNA of interest can be used as a delivery vehicle to deliver the DNA or RNA of interest to the target site of interest.

Modified megakaryocytes (or products thereof) expressing at least one peptide, polypeptide or protein of interest or containing DNA or RNA of interest can be used for the treatment of various diseases. shall be understood.

In some aspects of the present disclosure, the DNA or RNA of interest or the RNA or DNA encoding the protein of interest can be any gene that a skilled artisan desires to incorporate and/or express. In some embodiments, the at least one peptide, polypeptide or protein of interest comprises one or more nucleic acid molecules (i.e. genes of interest) encoding the at least one peptide, polypeptide or protein of interest. Expression in megakaryocytes can be achieved by delivery to spheres or progenitor cells, such as iPSC cells. In some embodiments, one or more nucleic acid molecules encoding at least one peptide, polypeptide or protein of interest may be contained within an expression vector. In some embodiments, vectors comprise one or more synthetic nucleotides (eg, locked nucleic acids, peptide nucleic acids, etc.) or nucleoside linkages (eg, phosphorothioate linkages). Vectors may be single stranded, double stranded, or contain regions that are both single and double stranded. Exemplary vectors include, but are not limited to, plasmids, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, herpes simplex viral vectors, poxviral vectors, and baculoviral vectors. be done. In some embodiments, a nucleic acid molecule encoding a peptide, polypeptide or protein of interest can also be expressed using a megakaryocyte-specific promoter. In some embodiments, the vector comprises a nucleic acid sequence encoding a therapeutic polypeptide or fragment thereof. In some embodiments, the vector includes a nucleic acid sequence that encodes the mRNA. In some embodiments, the vector comprises a therapeutic gene nucleic acid sequence, or fragment thereof, including a promoter. Vectors containing the nucleic acid molecule of interest can be transferred to cells (e.g., iPS cells, megakaryocyte progenitor cells) via any method known in the art, including, but not limited to, transduction, transfection, infection, and electroporation. cells, or megakaryocytes).

Targeted Delivery of Therapeutic Agents Encapsulated in Megakaryocytes and Platelets A therapeutic agent is loaded that is expected to be shielded from circulation when the product is transfused. Platelets are naturally recruited to cancer lesions, solid tumors, and circulating tumor cells, in some instances through receptor interactions with exposed collagen and other extracellular matrix components. , in other instances by receptors that are surface exposed upon platelet activation. Platelets are known to aggregate in response to tumor cells as a result of these interactions (this is also known as tumor cell-induced platelet aggregation). Platelets are 'activated' by these interactions, either by passive drug loading or by genetic modification of iPSCs, megakaryocyte progenitor cells, megakaryocytes, and any other cell that produces 'designer' platelets. These cells will secrete the contents of their secretory granules, which contain loaded small molecules and biotherapeutic agents. They also shed their membranes as part of the exocytotic process that produces microvesicles. In some embodiments, platelets covalently conjugated with small molecules and biological agents to the plasma membrane are expected to remain stably associated during microvesicle formation and are induced by tumor cells. It is selectively delivered to cancer as a result of platelet aggregation. In some embodiments, genetically modified human iPSCs, megakaryocyte progenitor cells, or megakaryocytes are treated with recombinant biological drugs fused to membrane-anchoring domains from surface receptors, in some instances CD3 and DAF. can be engineered to express and specifically deliver it to the cell surface. Recombinant biological drugs anchored to the plasma membrane can also be delivered to sites of disease pathology by incorporation into microvesicles as a result of platelet activation. Recombinant biological drugs anchored to the plasma membrane are also used in other examples of targeted drug delivery, such as enzymes, in some examples, solid tumors, cancer lesions, or sites of vascular injury or angiogenesis. A protease cleavage site may be included to allow matrix metalloproteases abundant at the site of disease pathology to cleave the recombinant biological drug.

The practice of the present disclosure may employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, unless otherwise specified, which are sufficient. is within the purview of those skilled in the art. Such techniques are described in "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", ( Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of this disclosure and, therefore, can be considered in making and practicing the disclosure. Techniques that are particularly useful for certain embodiments are discussed in the following sections.

Methods of Use As discussed above, in some embodiments, megakaryocyte progenitor cells, megakaryocytes, proplatelets, preplatelets, or platelets of the present disclosure are used for targeted delivery of such therapeutic compositions. can be modified to include a therapeutic composition for Specifically, megakaryocyte progenitor cells, megakaryocytes, proplatelets, preplatelets, or platelets of the present disclosure are conjugated (e.g., passively absorbed or covalently conjugated) either on or within their granules. It may be loaded with a therapeutic composition (by gating) or it may be genetically engineered to express a therapeutic composition.

In some embodiments, megakaryocyte progenitor cells, megakaryocytes, platelet precursors, preplatelets, or platelets of the present disclosure can be used in combination with other nanoparticulate materials for drug delivery. For example, in some embodiments, megakaryocyte progenitor cells, megakaryocytes, proplatelets, preplatelets, or platelet membranes of the present disclosure are adapted to interact with and transport therapeutic compositions. It can be used as an outer shell for drug delivery systems containing one or more materials that do. For example, the outer shell, the platelet membrane, may contain platelet proteins that are capable of interacting with cancer cells. In some embodiments, such drug delivery vehicles are used to lyse megakaryocyte progenitor cells, megakaryocytes, platelet precursors, preplatelets, or platelets of the present disclosure and to treat the outer membrane of the lysed cells. It can be prepared by filling the drug delivery system with the composition for use.

In some embodiments, megakaryocyte progenitor cells, megakaryocytes, platelet precursors, pre-platelets, or platelets of the present disclosure may be a source of growth factors, such as human growth factors. In some embodiments, such growth factors can be used for cell culture, tissue regeneration, wound healing, bone regeneration, functional cosmetics, and hemostatic bandages. In some embodiments, megakaryocyte progenitor cells, megakaryocytes, platelet precursors, pre-platelets, or platelets or lysates thereof or compositions thereof of the present disclosure can be used in cell culture. In some embodiments, the megakaryocyte progenitor cells, megakaryocytes, platelet precursors, pre-platelets, or platelets or lysates thereof or compositions thereof of the present disclosure can be used as functional cosmetics.

For example, platelets store bioactive factors acquired from megakaryocytes in secretory granules. Contents include platelet-derived growth factor isoforms (PDGF-AA, -AB and -BB), transforming growth factor-b (TGF-b), insulin-like growth factor 1 (IGF-1), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF or FGF-2), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF) and various chemokines and growth factors such as bone morphogenetic protein 2, bone morphogenetic protein 4 and bone morphogenetic protein 6 (BMP-2, BMP-4, BMP-6). Human platelet lysates dramatically increase ex vivo cellular expansion, improve bone marrow regeneration in vivo, and increase animal survival in radiological studies. In some embodiments, the present disclosure provides compositions or pharmaceutical compositions comprising the megakaryocyte-generated precursor platelets, pre-platelets or platelet lysates, such compositions comprising platelet-derived Growth factor isoforms PDGF-AA or PDGF-BB, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2), hematopoietic growth factor Flt3L, G-CSF , GM-CSF, interleukins (IL-1RA, IL-8, or IL-16), CXC chemokine family members CXCL1 (GRO alpha) or CXCL12 (SDF-1), TNF superfamily members sCD40L or TRAIL , or factors such as CC chemokine family members CCL5 (RANTES), CCL11 (eotaxin-1), CCL21 (6CKine) or CCL24 (eotaxin-2).

Administration Aspects of the present disclosure relate to pharmaceutical compositions comprising the present megakaryocyte progenitor cells, megakaryocytes, platelet precursors, preplatelets, or platelets according to embodiments of the present disclosure. In some embodiments, a pharmaceutical composition comprises the present megakaryocyte progenitor cells, megakaryocytes, platelet precursors, preplatelets, or platelets according to embodiments of the present disclosure together with a pharmaceutically acceptable carrier. For example, a carrier can be a diluent, adjuvant, preservative, antioxidant, solubilizer, emulsifier, buffer, water, aqueous solution, oil, excipient, adjuvant or vehicle, or a combination thereof. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin (Mack Publishing Co., Easton, Pa.); Gennaro, AR, Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington. In some embodiments, the carrier may be suitable for intravenous administration.

An aspect of the present disclosure is a method of treating a subject in need thereof, comprising administering the present megakaryocyte progenitor cell, megakaryocyte, platelet precursor, preplatelet, or platelet compositions to the subject in need thereof. It relates to a method comprising administering to the body.

Administration of a suitable dose and dosing regimen of the present megakaryocyte progenitor cell, megakaryocyte, platelet precursor, pre-platelet, or platelet-comprising compositions according to embodiments of the present disclosure to a subject in need thereof comprises: It can be determined based on age, sex, body weight, general medical condition, and the specific condition for administering the composition.

Megakaryocyte progenitor cells, megakaryocytes, platelet precursors, pre-platelets, or platelets according to embodiments of the present disclosure can be administered by any method. In some embodiments, the megakaryocyte progenitor cells, megakaryocytes, platelet precursors, preplatelets, or platelets can be administered by direct injection, eg, intravenous injection. Pharmaceutical preparations for injection can be prepared and delivered as known in the art.

Pharmaceutical Compositions This disclosure features methods for treating or preventing disease or infection in a subject. The invention also features a method for treating a wound. The method comprises administering to a subject in need thereof a therapeutically effective amount of a composition comprising induced pluripotent stem cell (iPSC)-derived platelets comprising a therapeutic agent. In embodiments, the compositions are used in pharmaceutical compositions.

In some embodiments, the pharmaceutical compositions described herein comprise a pharmaceutically acceptable carrier or excipient such as sterile water, saline solutions, buffered saline solutions, aqueous dextrose, aqueous glycerol, ethanol, or combinations thereof. The preparation of such solutions to ensure sterility, pH, isotonicity, and stability is performed according to protocols established in the art. Generally, a carrier or excipient is selected to minimize allergic and other undesirable effects and to be suitable for the particular route of administration, eg, subcutaneous, intramuscular, intranasal, and the like.

Administration of the pharmaceutical compositions contemplated herein can be carried out using conventional techniques such as, but not limited to, transfusion, transfusion, or parenterally. In some embodiments, parenteral administration is intravascular, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular , intrathecal and intrasternal infusion or injection.

Kits The disclosure provides kits comprising the megakaryocytes or differentiated cells of the disclosure. In one embodiment, the kit includes a composition comprising isolated megakaryocytes. In certain embodiments, the disclosure provides kits for differentiating, culturing, and/or isolating megakaryocytes or progenitors thereof of the disclosure. In certain embodiments, the present disclosure provides kits for producing platelets.

In some embodiments, the kit comprises a sterile container containing the cell composition, such container being a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other known in the art. other suitable container forms. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

If desired, kits are provided with instructions for generating megakaryocytes. The instructions generally include information regarding conditions and factors necessary for differentiation, culture, and/or isolation of megakaryocytes or progenitors thereof. In some embodiments, instructions for producing platelets are included. The instructions may be printed directly on the container (if present), may be printed as a label affixed to the container, or may be on a separate sheet, booklet, card supplied in or with the container. , or may be printed as a folder.

In the practice of this disclosure, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology are used and are within the skill of the art. Stay well within your capabilities. Such techniques are described in "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); Ｈａｎｄｂｏｏｋ ｏｆ Ｅｘｐｅｒｉｍｅｎｔａｌ Ｉｍｍｕｎｏｌｏｇｙ” （Ｗｅｉｒ， １９９６）；”Ｇｅｎｅ Ｔｒａｎｓｆｅｒ Ｖｅｃｔｏｒｓ ｆｏｒ Ｍａｍｍａｌｉａｎ Ｃｅｌｌｓ” （Ｍｉｌｌｅｒ ａｎｄ Ｃａｌｏｓ， １９８７）；”Ｃｕｒｒｅｎｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ” （Ａｕｓｕｂｅｌ， １９８７）；”ＰＣＲ： Ｔｈｅ Ｐｏｌｙｍｅｒａｓｅ Ｃｈａｉｎ Ｒｅａｃｔｉｏｎ”， （ Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of this disclosure, and thus can be considered in making and practicing the disclosure. Techniques that are particularly useful for certain embodiments are discussed in the following sections.

The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assays, screening, and therapeutic methods of the present disclosure, and are intended to assist the inventors in making their own It is not intended to limit the scope of what is considered a disclosure.

(Example 1)
Expansion of Clinical Grade hiPSCs Prior to differentiation, expansion of hiPSCs produces large numbers of cells for use in appropriately sized master and working cell banks, as well as cells suitable for clinical production. It is necessary to generate sufficient cell numbers to initiate differentiation at scale. Clinical grade hiPSC cell lines were obtained from the NINDS Human Cell and Data Repository (NHCDR) repository at the National Institute of Neurological Disorders and Stroke (NINDS)/NIH (National Institutes of Health). This cell line (NINDS ID: LiPSC-Gr1.1) was derived from male CD34 ⁺ cord blood (Lonza) and was treated with recombinant vitronectin (VTN) plus cGMP compatible reagents such as Essential 8, NutriStem. , or can be maintained and expanded in 2D culture using StemFlex (FIGS. 8A-8C). Characteristic colony growth and maintenance of pluripotent markers were observed in all three growth conditions (Figures 8A-8C, Figures 9A-9C).

Highly efficient single-cell passaging techniques have also been developed to support scale expansion of undifferentiated hiPSC cultures. The same methodology is also contemplated for banking and scale-up hiPSC seed trains leading to large-scale differentiation for clinical manufacturing. This approach provides rapid expansion for overall manufacturing capacity, undifferentiated pluripotent cultures with the ability to produce pre-MKs, and yields in systems compatible with cGMP manufacturing and clinical registration. and uniformity of culture performance. In this example, LiPSC-Gr1.1 cultures were dissociated into single cell suspensions using TrypLE (Thermo Fisher) followed by 0.5 μM H1152 (Tocris) and 10 ng/mL heregulin. Plated at defined densities in NutriStem hPSC XF (Biological Industries) containing β1 (Peprotech). Cultures were plated at a density of 1×10 ⁴ cells/cm ² at 4 day culture intervals and further plated at a density of 2×10 ⁴ cells/cm ² at 3 day culture intervals. . Cell attachment to untreated TC-flasks was mediated by 0.5% human AB serum (Valley Biomedical). The day after 18-22 hours after plating, cultures were fed with NurtriStem hPSC XF without supplementation. Cultures were passaged at intervals of 3 or 4 days to achieve predictable and consistent yields over multiple passages (Fig. 10A). The single-cell passaging mode also supports efficient cryopreservation in 10% DMSO (BloodStor100, STEMCELL Technologies), and cultures exhibit high thaw viability (>85%) and plating efficiency >100% adherent/viable/proliferating cells) when counted after 1 day (Fig. 10B). Thawed cultures showed >99% co-expression of SSEA-5 and TRA-1-60 cell surface markers of pluripotent cells (Fig. 10C), stained positive for Oct4 and Nanog (Fig. 13A), and spontaneously A lack of meaningful differentiation was confirmed.

To enable large-scale expansion, LiPSC-Gr1.1 cells were harvested from 2D cultures as single cells using TrypLE and placed in a stirred 3D vessel, in this case a 300 ml DasBOX mini bio. It was self-aggregated in a reactor system. For the first 24 hours, cells were supplemented with a ROCK inhibitor such as Y27632 to promote cell survival for 6-7 days during initial aggregation in a stirred tank. Within that time period, the resulting spheroids increased in diameter from 50 to 250 microns and the overall cell density increased up to 40-fold (Figures 11A-11C). hiPSCs grown in this manner were able to be repeatedly passaged to maintain their pluripotency for at least four consecutive rounds of expansion (FIGS. 12A-12B, FIG. 13B) and maintain a normal karyotype. (Fig. 14).

(Example 2)
Directed differentiation of hiPSCs into preMKs and MKs using a type IV collagen matrix in 2D culture vessels FIG. Differentiated into megakaryocytes using the 2D matrix-dependent directed differentiation protocol summarized in . A schematic diagram of the timeline shows each stage of differentiation (stages 0, 1, 2, 3), with the corresponding cell types and cell markers above the timeline, and media below the timeline. Composition, matrix, temperature and gas conditions are indicated.

PSCs, when harvested with 0.5 mM EDTA and plated as small clumps onto 4.2 ug/cm ² human collagen IV, exhibited a characteristic series of morphologies over the course of stage 1 differentiation for 6 days. (Fig. 15). At the end of Stage 1, representative wells are harvested as single cells using Accutase and assessed for hematopoietic endothelial markers CD31 and CD34 by flow cytometry (FIG. 16A). Across multiple independent iPSC differentiations (n=41), the average day 6 differentiation efficiency was approximately 40% CD31+ (range: ˜20-60%) and approximately 30% CD31+CD34+ (range: ˜15%-15%). 45%) (Fig. 16B).

Within 2-3 days after the onset of stage 2 (i.e., days 6+2 to 6+3), small round refractile cells appear within the adherent hematopoietic endothelial cells, eventually forming an adherent hematopoietic endothelial monolayer. released into the supernatant above (Fig. 17A). These released cells contain preMKs defined by cell surface expression of CD43 and CD41 and lack of expression of CD14 (FIGS. 17B, 17C). Floating and weakly adherent stage 2 cells that appear on top of these adherent cell layers are harvested daily by gently rinsing and collecting medium into conical tubes and analyzed daily for CD43, CD41, and CD14 expression. The purity of released cells is low in the first few days of stage 2, then plateaus, and by day 6+6 the average peak preMK purity is 50-60% (FIG. 18A). CD14+ myeloid cells are not a major contaminant of iPSC directed differentiation cultures during the first 6-7 days of stage 2, but there is some variation thereafter (Fig. 18B). The kinetics of preMK production peaked on average at days 6+6 and 6+7 and then declined (Fig. 19A). Across multiple independent iPSC differentiations (n=41), the average cumulative preMK (CD43+CD41+CD14−) yield was determined to be approximately 1 million per well (range: 100,000 to 3.3 million) (FIG. 19B). .

When preMKs from these cultures are transferred to stage 3 conditions, they differentiate into mature MKs within a few days. By days 2-4, the initial uniform small round refractile cells (Figure 20A) begin to increase in size (Figures 20B and 20C). At the same time, MKs producing platelet precursors can be readily observed (FIGS. 20C and 20D). By days 3-4 of stage 3, the percentage of CD61 ⁺ (megakaryocyte lineage) cells co-expressing the mature MK markers CD42a and CD42b was determined by FACS (FIGS. 21A and 21B), and mature MK (CD61+CD42a+CD42b+ cells ) can reach levels as high as 70-90% of all nucleated cells in culture (FIG. 21C).

(Example 3)
Directed Differentiation in Matrix-Independent 3D Cultures Differentiation from small-scale tissue culture plasticware (2D matrix dependent) into 3D scalable solutions to enable yields required for clinical production of megakaryocytes and platelets It is important to transition the entire process. An example of a scalable 3D solution involves performing differentiation using self-aggregating spheroids suspended in ultra-low attachment vessels that are being agitated or shaken (Fig. 3). In this example, LiPSC-Gr1.1 hiPSCs were dissociated into single cells using TrypLE and placed in pluripotent maintenance medium (e.g. Essential 8, Nutristem, StemFlex, other Resuspend at 500,000-1,000,000 cells/ml in similar media, or combinations thereof) in 6-well ultra-low attachment plates on an orbital shaker at 90 rpm or in spinner flasks with constant agitation ( Incubated at 37C, 5% CO2, 20% O2 in a 125 ml spinner flask (50 ml volume, 90 rpm). Within 24 hours in both systems, hiPSCs self-aggregated to form spheroids approximately 50-150 um in diameter (Figure 22A, see also Figures 23A and 11A for similar examples in different vessels). ). Agitation was then discontinued and the spheroids were allowed to settle to the bottom of the vessel (approximately 5 minutes). 50%-100% of the medium was then replaced with stage 1 differentiation medium to promote differentiation into hematopoietic endothelium, with agitation accompanied by incubation in hypoxic conditions (37C, 5% CO2, 5% O2). resumed. Medium changes were also performed daily for a total of 6 days (4 days at 37°C, 5% CO2, 5% O2, followed by 2 days at 37C, 5% CO2, 20% O2), during which time the spheroids became more It grew large and produced a characteristic structure and shape on day 6 (Fig. 22A). A sample of these spheroids at day 6 was dissociated and assessed by flow cytometry, and approximately 44% of the cells were found to express CD31 and CD34, markers of hematopoietic endothelium (Fig. 22B). , the purity compared favorably with 2D matrix-dependent cultures (Fig. 16B). To move to Stage 2, agitation was discontinued and the spheroids were allowed to settle to the bottom of the vessel (approximately 5 minutes). 50-100% of the medium was then replaced with stage 2 differentiation medium to facilitate differentiation and release of suspension cells (Figure 24A). Every day thereafter, suspension cells were harvested and partial medium changes were performed. To do this, agitation was discontinued and the hematopoietic endothelial spheroids were allowed to settle to the bottom of the vessel (approximately 5 minutes). Approximately 80% of the medium (with suspended cells) was collected and centrifuged. Half the working volume of fresh stage 2 differentiation medium was added to the spheroids along with a sufficient volume of conditioned medium (ie supernatant after centrifugation) to restore the original working volume. The remaining supernatant was then discarded and a portion of the cell pellet was used for FACS analysis (Fig. 24B) and the remainder either cryopreserved or transferred to stage 3 for maturation to mature MKs. Flow cytometric analysis of suspension cells showed that self-aggregated spheroids differentiated in 6-well ultra-low attachment plates on an orbital shaker and in 50 ml spinner flasks were similar. It was found to demonstrate both preMK production kinetics, purity and yield over time. Compared to 2D type IV collagen differentiation cultures, these 3D cultures produced more preMK at higher purity and early in stage 2 (FIGS. 24C, 24D).

Cultures expanded and harvested by single-cell passage (FIGS. 10A-10C) also aggregated into 3D spheroids and were effectively transformed into pre-MK cells using the 6-well suspension differentiation method as described. Differentiation was possible (Fig. 23A). Pre-MK yields were comparable to previous 6-well plate cultures, as was purity as assessed by co-staining for CD41/CD43 (FIG. 23B). These data demonstrate that hiPSCs effectively regulate self-renewal in single-cell passaging conditions, supporting stable expansion and scalability while retaining the ability to differentiate into pre-MK cells.

When transferred to stage 3 static culture, preMK from 3D self-aggregating spheroid cultures yielded similar MK purity as preMK from the 2D culture system (FIGS. 25A-25C). Furthermore, stage 3 differentiated cultures generated from 3D self-aggregating spheroid cultures increased dramatically in size, consistent with their identity as bona fide megakaryocytes, cells capable of generating platelet precursors. (Fig. 26).

(Example 4)
Addition of Soluble Laminin 521 During iPSC Aggregation or at Stage 1 to 2 Transition Improves Stage 2 preMK Yield in Two Different 3D Differentiation Modes Early iPSCs at 24 h (day −1) before initiation of differentiation Addition of laminin 521 during the aggregation step or when transitioning from stage 1 to stage 2 (day 6) increased preMK yield in two different 3D differentiation modes. 5000 single-cell dissociated iPSCs were dissociated per well of 96-well U-bottom ultra-low attachment plates containing StemFlex and ROCK inhibitor H1152 (control medium) with or without soluble recombinant laminin 521. sown. After 24 hours, the medium was replaced with stage 1 medium and medium changes were performed for 6 days. The medium was then replaced with stage 2 medium with or without soluble laminin-521. After 24 hours, half medium changes were performed daily for up to 6 additional days. Comparing preMK yields at stage 2 reveals that addition of laminin 521 on day -1 or day 6 of the differentiation process increased preMK yield compared to control cultures without laminin 521 addition. (Figs. 27A, 27B). To determine whether the effects of laminin-521 can also be observed in stirred 3D cultures, iPSCs were dissociated as single cells and 1.5 million cells were treated with StemFlex and ROCK with or without soluble recombinant laminin-521. Each well of a 6-well ultra-low attachment plate containing the inhibitor H1152 (control medium) was seeded and placed on an orbital shaker. After 24 hours, the medium was replaced with stage 1 medium and medium changes were performed for 6 days. The medium was then replaced with stage 2 medium with or without soluble laminin-521. After 24 hours, half medium changes were performed daily for up to 6 additional days. Comparing preMK yields at stage 2 reveals that addition of laminin 521 on day -1 or day 6 of the differentiation process increased preMK yield compared to control cultures without laminin 521 addition. (Fig. 27C). Laminin 521 action was amplified when iPSCs derived from the high-efficiency single-cell passaging technique described in Example 1 were similarly self-aggregated in NutriStem in 6-well plates on an orbital shaker (Fig. 27D).

(Example 5)
Adjusting the Order and Timing of Growth Factor Addition During Stage 1 Increases Differentiation Efficiency and Reduces Overall Growth Factor Usage The early specification events that occur during stage 1 of differentiation are complex. , requires a unique order and timing of cell signaling events. Therefore, adjusting the order and timing of the addition of the stage 1 media factors BMP4, bFGF and VEGFA compared to standard complete St1 media conditions that included all three growth factors throughout stage 1. This can improve the efficiency of the differentiation process and reduce growth factor usage. The first experiment (Experiment A) (Figure 28A) tested the order and timing of addition. When BMP4 alone was added for the first 24 hours, followed by VEGFA and bFGF (without BMP4) for the next 24 hours, followed by 4 days of complete St1 medium, preMK produced at stage 2 The numbers were significantly higher than control cultures that received complete St1 medium through stage 1 (FIG. 28B). Running the same sequence over 48 hours did not have the same effect. A second experiment (Experiment B) (Fig. 28C) demonstrates that BMP4 may be absent after 24 hours in this system, whereas FGF2 and VEGFA are important for the differentiation to proceed effectively. (Fig. 28D). This example demonstrates that stage 1 of differentiation can be effectively progressed using BMP4 alone for 24 hours followed by bFGF and VEGFA for 5 days before transitioning to stage 2 of differentiation.

(Example 6)
WNT Modulators Can Affect Stage 1 and Stage 2 Differentiation Efficiency WNT signaling is important during development. The GSK3 kinase inhibitors CHIR98014 and CHIR99021 act as WNT agonists. Only for the first 48 hours of differentiation, when the stage 1 differentiation conditions described herein were enhanced with 0.6 μM CHIR98014 or 6 μM CHIR99021, stage 1 differentiation as determined by immunofluorescent staining for CD31 and CD34 was reduced. A dramatic increase in differentiation efficiency was observed on day 6 (Figures 29A-29C). Control and CHIR98014 cultures were then transitioned to stage 2, where preMK production and release was followed by immunofluorescent staining for CD41 and CD43. Visual estimation of the number of CD41+ cells suggests that the higher stage 1 efficiencies produced by WNT modulators in the first 48 hours may correspond to higher production during stage 2 (FIGS. 30A-30B). . Therefore, short-term addition of WNT modulators can affect differentiation efficiency over subsequent stages of differentiation.

(Example 7)
Packed Bed Bioreactor Using Laminin 521-Coated Macrocarriers Here, laminin 521-coated PTFE macrocarriers in the shape of 1 mm Raschig rings can provide a support for iPSC differentiation and this macro The carrier material provides evidence demonstrating that it is expected to be suitable for use in a packed bed bioreactor as illustrated in the schematic shown in FIG. The PTFE rings were first incubated overnight with 1.25 ug/ml laminin-521 at 4° C. on a rocker. Prior to use, the PTFE rings were equilibrated in Essential 8 medium with the ROCK inhibitor H1152 in a 6-well plate. Pluripotent iPSCs were harvested using 0.5 mM EDTA, resuspended in Essential 8 medium with H1152, and seeded as clumps onto PTFE rings. Every 10 minutes the plate was shaken on an orbital shaker at 75 rpm for 30 seconds. After 1 hour, the plates were continuously shaken at 75 rpm overnight. After 24 hours, 90% of the medium was removed and replaced with Stage 1 medium, with daily medium changes. During stage 1, iPSCs exhibited growth areas on the interior of the Raschig rings (Fig. 31), which produced morphological features similar to those seen in 2D cultures (Fig. 22). Flow cytometric analysis of these cells showed a high percentage of hematopoietic endothelial cells, with approximately 80% cells expressing CD31 and more than half of these cells being double positive for CD34+. (Fig. 33A). Switching to Stage 2 medium and initiating half daily medium changes resulted in a change in morphology from generally flat colonies to 3D spheroid-type structures, but remarkably, these structures still remained intact. It adhered to the laminin 521 coating on the inside of the ring-shaped macrocarrier (Fig. 32). Cells released during stage 2 had high preMK content as early as day 6+2, approximately 75% of cells co-expressed CD43 and CD41 (Fig. 33B), and purity was 2D matrix dependent. It compared favorably with culture (Fig. 18A). Cells released on day 6+3 were harvested and cultured in stage 3 medium in ultra-low attachment plates for an additional 3 days, and approximately 80% of these cells co-expressed CD61 and CD42b. (Fig. 33C) shows that efficient MK differentiation occurred. Such macrocarriers allow the initial differentiation of iPSCs to hematopoietic endothelium (i.e. stage 1 of directed differentiation), plus further differentiation and release of preMKs (i.e. stage 2 of directed differentiation) in the same vessel. suitable for use as a material for a packed bed bioreactor capable of producing (Fig. 4). In this design, a packed-bed bioreactor is constructed using laminin-521-coated macrocarriers that are freshly seeded with pluripotent iPSCs. The packing layer may then be exposed to a continuous flow of medium, which allows stage 1 differentiation into hematopoietic endothelium. After infiltration of the packed bed, the medium will be circulated to the conditioning chamber where fresh medium components will be added and oxygen/CO2 concentrations will be adjusted via sparging or other means; The medium will then be recycled to the cells. Upon completion of stage 1, the medium will be switched to allow stage 2 differentiation and production and preMK release. Appropriately sized and shaped macrocarrier supports, e.g., 1 mm Raschig rings, provide sufficient medium to allow penetration through the packed bed of released cells and out of the reactor for collection and cryopreservation. expected to allow flow and channel width. This design reduces the shear forces experienced by the cells and, due to its perfusion-based design, allows for efficient media usage and continuous collection of preMKs as they are released.

(Example 8)
Detailed Characterization of iPSC-Derived Megakaryocytes The megakaryocytes generated using the methods described herein, when imaged by immunofluorescence microscopy, showed the MK-specific protein beta-1-tubulin ( 34), as well as proteins associated with alpha granules (PF4 and VWF, FIGS. 35A-35F) and proteins associated with dense granules (LAMP1 and serotonin, FIGS. 36A-36F), etc. associated with functional mature MKs. Demonstrate many peculiarities. Electron microscopy images of iPSC-derived MKs reveal characteristic ultrastructural features including multivesicular bodies, glycogen granules, and invaginated membrane systems (FIGS. 37A-37D). Gene expression analysis revealed downregulation of pluripotency genes such as OCT4 (Figure 38A) and upregulation of megakaryocytic lineage genes such as NFE2 (Figure 38B). A similar analysis was performed on a panel of related genes and the results of this analysis are consistent with the loss of the pluripotent stem cell signature and the gain of the megakaryocyte signature (Figure 39).

Compared to primary megakaryocytes (natural product) derived from bone marrow CD34 ⁺ , peripheral blood CD34 ⁺ , or cord blood CD34 ⁺ cells, iPSC-derived MKs were primary megakaryocytes derived from CD34 ⁺ bone marrow, peripheral blood, or cord blood stem cells. It had a similar average size (FIGS. 40A-40C) to megakaryocytes (natural product) (FIGS. 40C, 41B), but a lower characteristic ploidy distribution (FIGS. 41A-41B). ) was found. iPSC-derived megakaryocytes also have a characteristic growth factor, cytokine, and chemokine expression profile of factors similar to those present in human platelets, including the presence of multiple factors not previously reported in megakaryocytes. had (Fig. 42). To prepare the data, 25 million/mL hiPSC-MKs in 1×PBS were lysed by freezing cells at -80°C overnight and then thawing at 37°C. This freeze/thaw cycle was repeated four times. The resulting suspension was filtered using a 0.22 μm syringe filter. Lysates were tested for a select panel of growth factors, cytokines, and chemokines using multiplexed laser bead technology (Eve Technologies). Data were corrected for background (PBS, treated similarly to hiPSC-MKs), followed by commercial human platelet lysate (HPL), fresh MK differentiation medium (used at the final stage of differentiation), and conditioning. Compared to medium, MK differentiation medium removed from hiPSC-MKs prior to lysis. Although a strong overlap was observed between hiPSC-MKs and HPL, several proteins not previously described in megakaryocytes or platelets were also measured in hiPSC-MKs (Fig. 42).

The results described herein demonstrate a robust process for generating clinical grade human iPSC-derived megakaryocytes. Human iPSC-derived megakaryocytes can be isolated and enriched for further characterization or use in downstream applications such as the generation of human platelets.

(Example 9)
Detailed Characterization of Human iPSC-Derived Platelets Platelets are generated from human iPSC-derived mature MKs using the culture methods and harvesting techniques described in this application. Platelets may be harvested in static culture (Figs. 43A, 43B) or produced by feeding mature MKs to a millifluidics bioreactor at the apex of Stage 3 of the directed differentiation process (US9). , 795,965; US2017/0183616; US2018/0334652; see references in WO2018165308). Platelets elicited from the directed differentiation process stain negatively for DNA-intercalating dyes and are within a size distribution of 2-5 μm; pre-platelets are also observed with diameters of 5 μm and larger in this culture. (Fig. 43A). Human iPSC-derived platelets have a resting phenotype as indicated by micrographs showing distinct β1-tubulin rings and the absence of the activation marker CD62p (FIGS. 44B and 45A). These can be activated experimentally using glass diffusion techniques that reveal cytoskeletal changes indicative of filopodia and lamellipodia formation and spreading (FIGS. 44A and 44B).

Human iPSC-derived platelets have several features that distinguish them from primary donor-derived human platelets. They lack glycoprotein VI, a surface receptor abundantly expressed on human platelets (Figures 46A-46C). They also generate thrombin in greater amounts than platelets in plasma and over a shorter time frame after exposure to recombinant human tissue factor, a major initiator of the coagulation cascade (Fig. 47). Despite these differences, human iPSC-derived platelets show all indicators of functionality, including incorporation into thrombi formed in cremaster arterioles as part of a mouse model of laser-induced injury. (FIGS. 43A-43B, 44A-44B, 45, 46A-46C, and 47) are retained (FIGS. 48A-48C).

(Example 10)
Human iPSC-derived platelets take up recombinant biological agents by passive drug loading Platelets produced by the methods described herein, in addition to platelets extracted from whole peripheral blood, Platelets differentiated from blood cell sources have similar characteristics.

The line drawing in FIG. 6 enables drug loading into and onto preMKs, MKs, and PLCs by various methods, herein referring to any biologic, small molecule, or other form of therapeutic particle. provide an overview of what to do. In some forms, a millifluidics bioreactor (US9,795,965; US2017/0183616; US2018/0334652; see references in WO2018165308) is used to induce PLC production from MKs. The line drawing in FIG. 7 provides a schematic of the production of genetically modified versions of stem cells, in some forms iPS, ES, hematopoietic stem cells, etc., by lentiviral transduction of genetic material that integrates into the genome. In some embodiments, integration of genetic material into stem cells is accomplished using various nuclease-based techniques, homologous recombination, or other viral and non-viral methods. Integration of genetic material into the genome can occur in hematopoietic endothelium, preMK, and MK.

In one example, human IgG was loaded onto and/or onto washed donor-derived human platelets. Human IgG was conjugated to the NHS-ester Cy5.5 fluorophore at an 8-fold molar excess according to the manufacturer's instructions. The conjugated preparation was passed through a 40k molecular weight cutoff (mwco) zeba desalting column and quantified by a pierce 660 kit. The preparation was kept at 4 degrees Celsius until further use. For drug loading experiments, Cy5.5-conjugated human IgG was brought to room temperature and centrifuged at 15,000 rcf for 1 minute to remove aggregates. Antibodies were then added to 1×10e7 platelets in a reaction volume of 1 mL for 1 hour at 37 degrees Celsius. PGE1 was added at a final concentration of 1 ug/ml to inhibit platelet activation and cells were centrifuged at 1250 rcf for 17 minutes without brake. In preparations of platelets gated on CD61 expression (FIGS. 49A and 49B), this wash step was performed a second time to assess relative drug uptake after washing (FIGS. 49C and 49D). Drug uptake was visualized in cell pellets (Fig. 49E). A dose titration of human IgG was performed and flow cytometry shows that increasing input concentrations lead to concomitant increases in detectable human IgG (FIG. 49F). Mean fluorescence intensity was plotted as a function of human IgG dosage (Figure 49G) to quantify the amount of human IgG retained in washed platelets (Figure 49H).

Human donor platelets were able to take up concentrations of labeled anti-PD-L1 antibody (atezolizumab) up to 200 μg in subsequent experiments (FIG. 49I). 200 μg represents the therapeutic dose of atezolizumab. This experiment was designed to determine the maximal dose of uptake. However, platelets did not show a plateau of uptake or a maximal dose. To demonstrate that uptake was not antibody-specific, human IgG was labeled with CF55 (Biotium) as per manufacturer's instructions. Cells were fixed with 4% paraformaldehyde before fluorescence intensity measurements to prevent further uptake during analysis. Uptake of human IgG was monitored in a dose-dependent manner by measuring fluorescence intensity. These results do not represent the maximum concentrations allowed. After fixation, aliquots of the platelet preparations were washed and adhered to poly-l-lysine-coated glass coverslips by centrifugation. Platelets were then permeabilized with 0.5% Triton-X in PBS and blocked overnight with immunofluorescence blocking buffer (IFBB) (5 ml goat serum, 1% BSA in 50 ml PBS). Atezolizumab was visualized by incubation with AlexaFluor488 anti-human secondary IgG. Background fluorescence was monitored in samples exposed to atezolizumab or secondary antibody alone. For additional specificity, cells were labeled with CD61-APC (Biolegend). Coverslips were mounted on glass slides using Aqua-Poly/Mount (Fisher Scientific). Samples were imaged using a Zeiss Meta 880 confocal scanning microscope with Zen Black software for image acquisition (Fig. 50A). Image processing and analysis were completed using ImageJ software (Fiji/NIH). FIG. 50B is a high magnification of double-labeled platelets demonstrating subcellular localization within platelets.

Human iPSC-derived platelets generated using the methods described herein were loaded with the anti-CTLA4 antibody drug ipilimumab by co-incubation in aqueous buffer. Loading of ipilimumab at various concentrations resulted in flow cytometry histogram plots demonstrating a dose-dependent increase in encapsulated dose. Ipilimumab was conjugated to the NHS-ester Cy5.5 fluorophore at an 8-fold molar excess according to the manufacturer's instructions. The conjugated preparation was passed through a 40k molecular weight cutoff (mwco) zeba desalting column and quantified by a pierce 660 kit. The preparation was kept at 4 degrees Celsius until further use. For drug loading experiments, Cy5.5-conjugated ipilimumab was brought to room temperature and centrifuged at 15,000 rcf for 1 minute to remove aggregates. Ipilimumab was added to 1e6 human iPSC-derived platelets in a volume of 100 microliters. In one example, 1, 10, 30, and 60 μg of ipilimumab were added per sample, incubated for 1 hour at 37° C., washed and centrifuged to remove non-specifically bound drug. A flow cytometry-based histogram plot was generated showing a dose-dependent increase in ipilimumab encapsulation in human iPSC-derived platelets (Fig. 51A). In a separate experiment, ipilimumab not conjugated to Cy5.5 was loaded using the same technique onto human iPSC-derived platelets at a final concentration of 100 μg/ml, fixed with 4% paraformaldehyde, and immunofluorescence imaged. The cells were centrifuged onto poly-l-lysine-coated coverslips for cloning experiments. Platelets were then permeabilized with 0.5% Triton-X in PBS and blocked overnight with immunofluorescence blocking buffer (IFBB) (5 ml goat serum, 1% BSA in 50 ml PBS). Platelets were stained with granules (PF4) and surface (CD61) markers and drug signals were found to be intracellular specks. The resulting micrographs demonstrate ipilimumab loading on CD61+ human iPSC-derived platelets, including evidence of ipilimumab localization on the cell surface and within selected alpha granules, as delineated by PF4 staining (Fig. FIG. 51B).

A further example of recombinant protein loading into platelets was performed on CD34+-derived material using ipilimumab conjugated to the fluorophore Cy5.5 using the methods described herein. CD34+ derived platelets were analyzed for size and granularity (Figure 52A) and CD61 expression (Figure 52B) to confirm the platelet phenotype. When ipilimumab was incubated with platelet preparations at various concentrations for 1 hour at 37 degrees Celsius, the maximal signal established at 600 μg/ml ipilimumab was not seen (Fig. 52C). Measured values were converted to pg of ipilimumab per platelet in the reaction vessel, and an input ratio of approximately 100-300 pg/PLC was sufficient to see maximal signal by flow cytometry (Fig. 52D). Final concentrations of retained ipilimumab per platelet were found to be between 1 and 6 pg/platelet, depending on treatment dose (FIG. 52E). After fixation, aliquots of the platelet preparations were washed and adhered to poly-l-lysine-coated glass coverslips by centrifugation. Platelets were then permeabilized with 0.5% Triton-X in PBS and blocked overnight with immunofluorescence blocking buffer (IFBB) (5 ml goat serum, 1% BSA in 50 ml PBS). Platelets were stained with granules (PF4) and surface (CD61) markers and drug signals were found to be intracellular specks (Fig. 52F).

To determine whether donor-derived human platelets take up atezolizumab (FIG. 53A) or ipilimumab (FIG. 53B) and remain functional, platelets were incubated with 15 μg of Dylight-488 labeled antibody for 30 minutes. Identical samples were then exposed to human thrombin (1 unit/ml) for the last 10 minutes of a 30 minute incubation. To prevent further uptake during analysis, cells were immediately fixed with 4% paraformaldehyde and analyzed for mean fluorescence intensity by FACS and compared to samples loaded with drug alone (unconjugated). As observed in Figures 53A and 53B, platelet activation reduced fluorescence intensity as seen by the shift of the labeled drug histogram to the left (closer to drug alone). FIG. 53C is a resting platelet loaded with fluorescent PDL1 (as in 13A). FIG. 53D is an activated platelet loaded with PDL1, but when glass-activated, it no longer contains fluorescent PDL1. These data suggest that platelet activation with thrombin causes antibody release and demonstrate that drug-loaded platelets remain functional and are capable of release upon agonist stimulation.

(Example 11)
Covalent Conjugation of Recombinant Proteins in Human iPSC-Derived Platelets There are many strategies for creating covalent linkages of recombinant proteins onto cell membranes (FIG. 6). For the iPSC-derived platelets described herein, 2-iminothiolane (Traut's Reagent, Thermofisher #26101) at various concentrations was incubated with 1e6 platelets in 500 ul of buffer at 37 degrees Celsius for 1 hour. to convert primary amines to sulfhydryls (depicted in FIG. 54A). At the same time, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, Thermofisher #22360) was incubated with human IgG for 2 hours at 4C (depicted in Figure 54B). Platelets treated with Traut's reagent were then incubated with SMCC linker-linked IgG for 1 hour at 37 degrees Celsius (Fig. 54C). Trout dose titrations were performed on washed donor platelets using a fixed concentration of SMCC linker-linked IgG (versus IgG without SMCC). By flow cytometry, the signal was significantly greater with increasing doses of Traut's reagent for SMCC linker-ligated IgG, with maximal efficiency observed at 0.4 mg/ml Traut's reagent ( Figure 54D). Without the SMCC linker, the IgG signal was not increased by flow cytometry (Fig. 54E), indicating that the conjugation reaction was efficient in promoting IgG conjugation to the surface of washed platelets. It is suggested that the

The protocol described in Figures 54A-54C was then used on human iPSC-derived platelets using anti-CTLA4 commercial antibody ipilimumab (Selleckchem #A2001). Ipilimumab-conjugated platelets retained CD61 expression (FIG. 55A) and were not activated by the procedure as assessed by CD62p expression (data not shown). By flow cytometry (gated on CD61 expression) using SMCC linker-linked ipilimumab (Fig. 55B, indicated by an arrow) with platelets treated with Traut's reagent, ipilimumab was at the highest abundance. observed. The same cells were fixed in 4% PFA, mounted on poly-l-lysine-coated coverslips, and then stained for CD61 and PF4 using the methods described herein. Ipilimumab was observed to be mostly conjugated on the cell surface (Fig. 55C).

(Example 12)
Passive loading of human iPSC-derived preMKs and MKs with recombinant drug biologics. Induced pluripotent stem cells were differentiated into mature megakaryocytes using the methods described herein. Cells were incubated with Dylight 488-labeled atezolizumab for 30 minutes, fixed with 4% paraformaldehyde, and centrifuged onto prepared poly-l-lysine-coated glass coverslips. Cells were additionally stained for CD61 to confirm mature megakaryocytes in culture. CD61 was visualized using a pre-conjugated CD61-APC antibody. Samples were washed and mounted on glass coverslips using Aqua-poly/mount (Fisher Scientific). Images were captured using a Zeiss Meta 880 confocal scanning microscope and analyzed using Fiji ImageJ software (Figures 56A-56C). These data suggest that the megakaryocytes of the present disclosure and their generated resting platelets are capable of taking up atezolizumab. Atezolizumab was demonstrated to co-localize with alpha granule-stained PF4 (Fig. 56C) and is also shown to be on the cell surface. This suggests that passive loading (as opposed to covalent conjugation to the cell membrane) can facilitate the localization of alpha granules in platelet-producing MKs, and that during platelet differentiation the granules provide evidence that it can be retained in

Megakaryocyte progenitor cells, preMK, were loaded with an unconjugated version of the anti-CTLA4 antibody, ipilimumab, using 1e6 cells in 1 ml and 100 ug of ipilimumab. preMKs were fixed on poly-1-lysine-coated coverslips and stained with fibrinogen (alpha granule stain) and CD61 (surface marker) (FIG. 57). Since ipilimumab was observed to co-localize with both CD61 and fibrinogen, preMKs could be loaded with antibody drugs, potentially loading the drug into the granules throughout the differentiation process to MKs and platelets. It is suggested that it can be held.

(Example 13)
Covalent Conjugation of Recombinant Drug Biologics to Human iPSC-Derived PreMKs and MKs The ability to conjugate is demonstrated herein. preMKs were harvested from stage 2 cultures and immunophenotyped for CD41 and CD43 co-expression (Figures 58A and 58B). iPSC-derived preMKs were treated with 0.4 mg/ml Traut's reagent with 1e6 cells in 500 ul buffer for 1 hour at 37° C. to convert primary amines to sulfhydryls. Simultaneously, SMCCs were incubated with ipilimumab for 2 hours at 4°C. Traut's reagent-treated preMK was then incubated with 100 μg/ml SMCC linker-linked ipilimumab for 1 hour at 37°C. A secondary antibody against human IgG conjugated to AlexaFluor 647 was used to detect conjugated ipilimumab. In the absence of drug treatment, no drug was detectable in cells that were double positive for CD41 and CD43 (Fig. 58C). For drug-treated samples, all observable preMKs double positive for CD41 and CD43 had detectable ipilimumab (FIG. 58D). Mature megakaryocytes were harvested at stage 3 of a directed differentiation protocol from human iPSCs and immunophenotyped for CD61 (Figure 59A) and CD42a (Figure 59B). MKs were treated with 1e6 cells in 500 μl buffer with 0.4 mg/ml Traut's reagent for 1 h at 37° C. to convert primary amines to sulfhydryls. Simultaneously, SMCCs were incubated with ipilimumab for 2 hours at 4°C. SMCC linker-linked ipilimumab was reacted with trout-treated MK at 37° C. for 1 hour. A secondary antibody against human IgG conjugated to AlexaFluor 647 was used to detect conjugated ipilimumab. In the absence of drug treatment, no drug was detectable in cells that were double positive for CD61 and CD42a (Fig. 59C). For drug-treated samples, all observable MKs double positive for CD61 and CD42a had detectable ipilimumab (FIG. 59D).

This data demonstrates the covalent conjugation of recombinant proteins, biological agents, to human induced pluripotent stem cell (iPSC)-derived megakaryocyte progenitor cells (preMK) and mature megakaryocytes (MK). .

(Example 14)
Small molecule loading of washed human platelets by passive diffusion. Sigma #D1515). Genomic material would typically be expected to sequester the drug within the cell as a result of co-incubation with a platelet preparation and entry into the cell by passive diffusion, since platelets are a nucleate-free cell type. , does not contain such genomic material. Doxorubicin at 100 μM was used with a preparation of 1e7 platelets in 1 ml of buffer, in a dialysis cassette (Thermofisher #88400) under constant agitation on an orbital shaker at ambient temperature for 30, 120, 240, and incubated for 1440 minutes. Doxorubicin has unique fluorescent properties that can be detected by flow cytometry (Ex 427 nm/Em 585 nm), allowing multiple washing steps to be performed on platelet preparations and non-specifically bound It was found that the drug cargo is expected to be retained even after the molecules are gone (Fig. 60A). A kinetic study was employed to understand the minimum and maximum time required for doxorubicin encapsulation into washed platelets. It was observed that 30 minutes was sufficient to see detectable doxorubicin expression in sampled platelets and the signal was retained after 1440 minutes (Fig. 60B). This data suggests that small molecule drugs can be efficiently trapped in platelets.

(Example 15)
Generation of Platelets from Genetically Modified Megakaryocyte Progenitors Platelets expressing therapeutic transgenes are expected to represent a significant advance in the treatment of injury, disease, and disease. To generate transgene-expressing platelets, megakaryocyte progenitors were transduced with a lentiviral vector containing a nucleic acid cassette encoding a reporter protein. Specifically, this cassette encoded the EF1 alpha promoter and ZsGreen fluorescent protein. 42 hours after infection with lentiviral vectors, fluorescence was detected in transduced megakaryocyte progenitors, but not in untransduced (mock) controls (Fig. 61), indicating that megakaryocytes Progenitors are shown to be successfully transduced.

Transgene-carrying megakaryocyte progenitors were cultured according to the methods described herein to produce platelets. Referring to FIG. 62A, CD61+ cells (i.e., platelets) derived from transduced megakaryocyte progenitors showed expression of the reporter protein, whereas CD61 cells from mock transduced megakaryocyte progenitors showed expression of the reporter protein. No fluorescence was visible in plucked CD61 cells, indicating that the nucleic acid cassette was successfully inherited from transduced megakaryocyte progenitors. To verify that the fluorescent signal was caused by platelets, platelets elicited from mock- and lentiviral-transduced megakaryocytes were sorted using a CD61 gating strategy (Fig. 62B). The fluorescence histogram shown in Figure 62C demonstrates that fluorescence signals were detected in CD61+ platelets.

(Example 16)
Platelet Production Using a Bioreactor System A bioreactor and conditions suitable for therapeutic agent loading or genetic modification were used to produce platelets. To produce platelets, megakaryocytes were seeded into a platelet bioreactor (Fig. 63A). The bioreactor consisted of two continuous channels (FIGS. 63B, 63C) separated by a porous membrane (5 μm pores) (FIG. 64). Megakaryocytes were immobilized on the porous membrane with high efficiency (>99% retention as demonstrated by flow cytometric analysis), and the medium flowing in the channels provided a physiologically relevant A shear force is applied (Fig. 64). Independently adjusting the flow rates in the first channel and the second channel to selectively trap megakaryocytes on the membrane and further along the second channel on the trapped megakaryocytes. Physiological shear rates are created by creating a differential between the configured channels, causing platelets to be produced.

Megakaryocytes immobilized on the porous membrane extend processes into the second chamber, which causes the megakaryocytes to release platelets (FIGS. 64 and 65), thereby releasing platelets into the second channel. (Fig. 64). Because bioreactors mimic the physiological conditions during platelet formation (eg, the shear forces of the bone marrow environment), bioreactor-produced platelets are functional and provide a surrogate for human platelets. Platelets produced using the bioreactor methods described herein can be used for transfusion and drug delivery.

(Example 18)
Generation of Platelets from Genetically Modified iPSCs iPSCs, including the iPSCs described herein, were genetically modified using a replication-incompetent lentivirus (as outlined in FIG. 7) to transform them with green fluorescent protein. A ZsGreen was expressed. ZsGreen+ cells at each differentiation stage, such as iPSC populations, stage 2 pre-MK populations, stage 3 induced MK populations, and PLC products generated from the Millifluidics bioreactor described herein was observed (Fig. 66A). iPSCs were lentivirally transduced and observed to contain ZsGreen+ cells by fluorescence microscopy as early as 48 hours post-transduction. These cells were then separated into single-cell derived clones and the resulting ubiquity of ZsGreen expression was observed by fluorescence microscopy and represented graphically in FIG. 66B. Cell expansion (ie stage 1) was performed according to the protocol described in Example 1 using the 3D system (see Example 3). ZsGreen expression was observed to be retained in cell types validated to be pre-MK during separate time points in stage 2 of differentiation (FIG. 66C). Megakaryocytes (MKs) were observed to retain a high degree of ZsGreen signal as verified by their CD61 and CD42b dual expression, representing approximately 77% of the cell cultures in this example ( Figure 66D). After seeding MKs in a millifluidics bioreactor and generating and purifying PLCs, the PLC samples that were first gated by flow cytometry for CD61 expression were, in this example, ZsGreen protein translated in MK cultures. Approximately 24% of the objects were retained (Fig. 66E).

(Example 19)
Comparison of PLC surface expression of platelet-specific markers and aberrant signaling pathways compared to donor-derived platelets.
PLC and donor-derived platelets were compared. Flow analysis of cell surface markers revealed a unique surface profile with numerous characteristic markers of platelets.

FIG. 67 depicts representative flow cytometry scatter plots from freshly isolated donor platelets and PLCs produced by the Millifluidics bioreactor. A representative donor sample contains a tight population of DRAQ5 events and calcein AM positive cells (FITC channels). The Calcein AM FITC fluorescent probe is applied to the sample in a quenched configuration and becomes cleaved by cellular esterases upon entry into intact cells. Calcein AM positive events represent intact cells (platelets). Donor platelets display a uniform signal (FITC intensity). A representative PLC scatter plot demonstrates a larger range size due to forward and side scatter. The size distribution of DRAQ5 events corresponds to the unlabeled scatter plot. Unlike donor platelets, a large population of cells are not labeled with Calcein AM.

FIG. 68 depicts representative flow cytometry scatter plots from freshly isolated donor platelets and PLCs produced by the Millifluidics bioreactor. Cell populations are gated for DRAQ5-negative events before CD61+ (VioBlue events). In the donor platelet population, the entire population is CD61 positive (GPIIIa), the major platelet identification marker, compared to a distinct CD61− population in PLC. The platelet activation dependent marker CD62p was monitored in each sample for activation status. Resting conditions did not significantly activate any cell population.

Figure 69 shows a comparison of elongated surface markers of donor platelets versus PLC. It was demonstrated that there was little difference in CD42a surface expression between donor platelets and PLCs. CD42b (glycoprotein Ib), a major member of the complex forming the receptor for von Willebrand factor, was found to be significantly reduced in PLC when compared to donor platelets. The plot also depicts a significant reduction in CD36 (glycoprotein IV) in PLCs when compared to donor platelets, CD36 being a major mediator of the early events of platelet adhesion to collagen and the release of thrombospondin. serves as a receptor. These analyzes confirm a unique surface phenotype for in vitro-induced, bioreactor-produced PLCs compared to normal adult donor samples.

Furthermore, analysis of activated PLC compared to adult donor platelets demonstrated different biological responses to agonists. Figure 70 compares the platelet activation marker CD62p after exposure to thrombin receptor activator peptide (TRAP-6). Compared to the significant shift in surface marker expression in donor platelets, PLC demonstrated little change after exposure to agonist. Figure 71 compares the platelet activation marker CD62p after exposure to thrombin (1 U/ml). Compared to the significant shift in surface marker expression in donor platelets, PLC demonstrated no change after exposure to strong platelet agonists. Differences in biological responses to platelet-activating factor confirm that PLCs indicate altered cell signaling networks to donor platelets, suggesting that the in vitro-induced product distinct Emphasize characteristics.

(Example 20)
The composition and function of PLC differs from donor platelets.
Analysis of different particle sizes and proportions within the PLC product using a number of approaches indicated the presence of a large population of microparticles. FIG. 72 depicts a representative histogram generated using the Multisizer device. Donor platelets show a peak size in % of approximately 2.5 μm in size. Although PLCs contain cells to a similar extent, PLCs contain a significant percentage of cells and microvesicles smaller than 2 μm. Equivalent numbers of CD61+ platelet particles were analyzed in each sample.

Figure 73 shows representative forward and side scatter plots of donor platelets and PLC. The plot depicts a substantially higher proportion of microvesicle size events in PLC compared to donor platelets. Control samples were used in both the Multisizer and flow cytometry experiments to demonstrate that the microparticle content of the PLC samples was derived from the cellular components of the bioreactor process (MKs and platelets) and not from the cell culture medium. confirmed. Figures 74A and 74B compare the morphology of platelets (Figure 74A) versus PLC (Figure 74B) by donor electron microscopy, scale bars are placed within the micrographs. Although platelet morphology is comparable in each sample, Figure 74B contains an increased number of microvesicles (bottom left quadrant). Figure 75 shows microvesicles derived from iPSC-derived MKs and within the PLC population. They are heterogeneous in size from less than 40 nm in diameter to over 1000 nm.

Figure 76 shows the reproducibility of thrombin generation in PLC compared to donor platelets. Each group represents three independent samples monitored for thrombin generation as described in Methods. In each example, the PLC showed a shorter lag time to peak thrombin generation in addition to a larger peak in thrombin generation. FIG. 77 depicts representative dose and response curves for PLC in thrombin generation. Altering the cell density of the PLC (0.5×10 ⁶ to 3.0×10 ⁶ ) and the associated increase in microparticle content lead to increased peak thrombin generation with little change in lag time.

Figure 78 depicts the contribution of microvesicles to thrombin generation within the PLC. As shown in Figure 76, PLC showed a rapid and strong burst of thrombin generation unlike that of fresh donor platelets. In FIG. 78A, the PLC was subjected to a 1 μm filter to remove all cellular material larger than 1 μm. As shown in Figure 78B, filtered PLC microparticles) exhibited thrombogenic activity resulting in thrombin levels similar to those of donor platelets. The filtered product was evaluated by TGA using a sample volume equal to the prefiltered sample. This was not adjusted for microparticle count and thus was substantially underloaded compared to the prefiltered sample. These size, ultrastructural and particle functionality analyzes indicate that PLC is an important population of cell-derived microvesicles that may provide a substantial contribution to the thrombogenic potential of bioreactor-derived products. It was demonstrated to show that In addition, these microparticles are derived from process megakaryocytes and platelets, and potentially growth factors, cytokines, wound healing factors and other biological properties with commercial significance, as well as designer payload delivery. may be one of the most attractive sources of potential vehicles for future therapeutic agents that utilize them.

The results reported in Examples 19 and 20 above were obtained using the following materials and methods.

Preparation of Donor Platelets Whole blood was collected and centrifuged at 150 xg for 17 minutes without brake. Approximately 2 mL of Platelet Rich Platelet (PRP) was then removed and placed in a 15 mL conical tube. PRP was diluted with a 1:2 dilution of platelet wash buffer and a 1:10000 dilution of PGE ₁ (prostaglandin E1). PRP was centrifuged at 460 xg for 17 minutes with the brake set to 3. Cells were resuspended in 1 mL platelet additive solution (PAS) and supplemented with a 1:2 dilution of platelet wash buffer/1:10000 dilution of PGE1. Samples were centrifuged at 460 xg for 17 minutes with the brake set to 3. Cells were resuspended in PAS and concentrations were calculated according to readouts taken from MACSQuant Analyzer 10 (Miltenyi).

Bioreactor Product Concentration and Medium Exchange Platelets were collected from the bioreactor in medium and a 1:10000 dilution of 1 mg/mL PGE ₁ was added just prior to concentration. Platelets were centrifuged at 1250 xg for 30 minutes with the brake set to 3. The pellet was resuspended in 1 mL PAS. Centrifugation was repeated as a wash step with the addition of a 1:10000 dilution of 1 mg/mL PGE ₁ again just prior to spinning. Cells were resuspended and left at 37°C until further processing.

Staining of Platelets (DRAQ5, Calcein AM, CD61, CD42a, CD42b, CD36, CD62p) Known platelet-specific surface markers DRAQ5 nuclear pigment (Cell Signaling Technologies #4084L) and CD61 (VioBlue, Miltenyi #130-110-754) Platelets/PLC were counted using flow cytometry with staining for ). Platelets were designated as either DRAQ5-negative and CD61-positive events. Also includes Calcein AM (BioLegend #425201), CD42a (PE, Miltenyi #130-100-966), CD42b (PE, Miltenyi #130-100-199), and CD36 (PE, Miltenyi #130-095-472) Platelets were stained with additional markers to further assess cell integrity. All samples were acquired on a MACSQuant Analyzer 10 (Miltenyi) and analyzed using FlowJo software. The gating strategy was based on forward and side scatter parameters determined by appropriate IgG controls.

Activation of Samples by TRAP6 and Thrombin Platelets were assessed by flow cytometry for their ability to activate in response to two agonists, TRAP6 (Bachem #4031274) and thrombin (Enzyme Research Laboratories, #HT1002a). Platelet samples were incubated with agonists for 10 minutes at 37°C. Additional platelet samples were incubated under the same conditions without agonist to determine baseline activation. Samples were then stained for DRAQ5 and CD61 as described above. Platelets were also stained for 15 minutes for CD62p (PE-Vio770, Miltenyi #130-120-725), a standard marker of activation. Activation was quantified by changes in CD62p expression of DRAQ5-negative, CD61-positive events between baseline and agonist-induced conditions. All samples were acquired on a MACSQuant Analyzer 10 (Miltenyi) and analyzed using FlowJo software. The gating strategy was based on forward and side scatter parameters determined by appropriate IgG controls.

Multisizer Particle sizing was performed using Beckman's Multisizer 4 using an aperture of 0-12 μm, according to the manufacturing protocol. Prior to measurement, samples were filtered using a 15 μm filter to prevent clogging. Measurements were performed using isoton II buffer.

Electron Microscopy Platelets were placed in 2.5% formaldehyde, glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, and pelleted at 1500 xg for 15 minutes. The supernatant was removed and replaced with 1 mL of 0.1 M sodium cacodylate. Samples were stored at 4° C. until further processing by the Harvard Medical School Electron Microscopy Core (Boston, Mass.). Ultrathin sections were stained with uranyl acetate and lead citrate and examined on a Tecnai G2 Spirit BioTWIN transmission electron microscope at an accelerating voltage of 80 kV. Images were recorded using an AMT 2k CCD camera and accompanying proprietary in-house software.

Thrombin generation assay (TGA)
The platelet/PLC concentration was adjusted to approximately 10x the density required for the assay. Microparticles were removed from George King Pooled Normal Plasma (GK-PNP) control samples by spinning at 20,000 xg for 30 minutes in a temperature-controlled microcentrifuge. Platelet samples were resuspended in GK-PNP to a final concentration of 5×10 ⁷ platelets/mL. TGA Reagent, Reagent C Low and TGA Substrate were prepared according to the manufacturer's instructions. The plate reader was set as directed to ensure a temperature of 37°C. Samples and controls were measured in duplicate. Thrombin generation is quantified by enzymatic conversion of a thrombin substrate into a fluorogenic molecule that can be detected by standard methods.

OTHER EMBODIMENTS From the foregoing description, it will be apparent that variations and modifications can be made to the disclosure herein to adapt it to various uses and conditions. Such embodiments are also within the scope of the following claims. In particular, various variations useful in the methods and compositions of this disclosure are disclosed in commonly owned PCT/US2019/012437 filed Jan. 5, 2019, as well as US9,763,984, US9, 795,965; US2017/0183616; US2018/0334652; WO2018165308, all of which are hereby incorporated by reference in their entireties.

A listing listing of elements in any definition of a variable herein includes the definition of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

All patents and publications referred to in this specification are herein incorporated by reference as if each independent patent and publication were specifically and individually indicated to be incorporated by reference.

Claims (17)

A composition comprising a population of induced pluripotent stem cell (iPSC)-derived platelets, wherein said platelets exhibit increased thrombin generation compared to a donor-derived platelet population having similar cell densities. 2. The composition of claim 1, wherein said thrombin generation in said iPSC-derived platelet population is 2-3 times greater than said thrombin generation in said donor-derived platelet population. 2. The composition of claim 1, wherein said thrombin generation results in a relative concentration of thrombin of about 250 to about 600 nM for a cell density of about 0.5 x ¹⁰⁶ to 3.0 x ¹⁰⁶ . 4. Any of claims 1-3, wherein the lag time between stimulation and achieving maximum thrombin concentration in the iPSC-derived platelet population is reduced compared to the lag time for the donor-derived platelet population. or the composition according to claim 1. wherein said lag time for achieving maximum thrombin concentration in said population of induced pluripotent stem cell-derived platelets is about 7 to 13 minutes, and said population contains about 0.5 x ¹⁰ platelets/mL each; to 3×10 ⁶ platelets/mL. 4. The composition of any one of claims 1-3, wherein the iPSC-derived platelet population comprises platelets that do not express glycoprotein VI. said iPSC-derived platelet population from a CD61+, DRAQ-, calcein AM+, CD42a+, and CD62P- biomarker profile; a CD61+, DRAQ-, calcein AM+, CD42a+, and CD62P+ biomarker profile; 4. The composition of any one of claims 1-3, comprising platelets having a biomarker profile selected from the group consisting of: 8. The composition of claim 7, wherein the iPSC-derived platelet population comprises platelets that do not express glycoprotein VI. 4. The composition of any one of claims 1-3, wherein greater than 70% of said iPSC-derived platelets express CD61+. 4. The composition of any one of claims 1-3, wherein less than 10% of the iPSC-derived platelets express CD42b. 4. The composition of any one of claims 1-3, wherein less than 5% of the iPSC-derived platelets express CD36 (glycoprotein IV). 4. The composition of any one of claims 1-3, wherein less than 35% of the iPSC-derived platelets express calcein. 4. The composition of any one of claims 1-3, wherein the iPSC-derived platelet population has altered cell signaling compared to donor-derived platelets or megakaryocytes. 14. The method of claim 13, wherein said altered cell signaling comprises reduced CD62 activation following exposure to TRAP-6 or thrombin, or wherein less than 15% of said iPSC-derived platelets contain CD62p. Composition. 4. The composition of any one of claims 1-3, further comprising thrombogenic microparticles such that the composition has a peak size of less than approximately 2 [mu]m. 4. The composition of any one of claims 1-3, wherein the thrombogenic microparticles range in size from 40 nm to 100 nm in diameter. 17. The composition of claim 16, wherein said thrombogenic microparticles constitute more than 50% of said composition.

Images